WO2004070058A1

WO2004070058A1 - Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of copd

Info

Publication number: WO2004070058A1
Application number: PCT/EP2004/000542
Authority: WO
Inventors: Jeffrey Encinas; Shinichi Watanabe; Kevin Bacon
Original assignee: Bayer Healthcare Ag
Priority date: 2003-02-03
Filing date: 2004-01-23
Publication date: 2004-08-19

Abstract

Human membrane spanning 4-domains, subfamily A, member 8B (MS4A8B) is overexpressed in COPD patients and is useful as a diagnostic marker and target for treatment. Methods are disclosed for predicting, diagnosing and prognosing as well as preventing and treating COPD with the use of MS4A8B. Reagents which regulate human MS4A8B can play a role in preventing, ameliorating, or correcting dysfunctions or diseases such as chronic obstructive pulmonary disease. RCK 45-Foreign Countries - 2 -RCK 45-Foreign Countries Sp/ngb/NT- 1 -

Description

METHODS AND COMPOSITIONS FOR THE PREDICTION, DIAGNOSIS, PROGNOSIS. PREVENTION AND TREATMENT OF COPD

TECHNICAL FIELD OF THE INVENTION

The invention relates to methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of chronic obstructive pulmonary disease (COPD). The invention is based on the discovery that the gene of human membrane spanning 4-domains, subfamily A, member 8B (MS4A8B) is overexpressed in COPD patients. The invention discloses that MS4A8B is useful as a diagnostic marker and target for treatment. Methods are disclosed for predicting, diagnosing and prognosing as well as preventing and treating COPD with the use of MS4A8B.

TECHNICAL FIELD OF THE INVENTION

'Membrane spanning 4- domains gene family

Membrane spanning 4-domains, subfamily A (MS4A subfamily) is a protein class having four putative transmembrane regions (Tedder et all J Immunol 141, 4388-4394, 1988). CD20, the high-affinity IgE receptor β chain (FcεRIβ), and HTm4 are representative members of the MS4A subfamily of proteins, and are all structurally related cell surface proteins expressed by hematopoietic cells. Currently, 24 human, mouse, and pig genes have been identified in the protein family and can be subdivided into at least 12 groups (MS4A1-MS4A12). Like CD20 and FcεRIβ, the 9 other human MS4A family members are likely to be components of oligomeric cell surface complexes involved in signal transduction in diverse cell lineages. CD20 forms a homo- or perhaps heterotetrameric complex that regulates Ca²⁺ conductance by either forming or serving as a functional component of a Ca²⁺-permeable cation channel (Kanzaki et al, J Biol Chem 272:14733-14739, 1997). FcεRIβ is a component of a tetrameric receptor complex consisting of α, β, and two γ chains. FcεRI mediates interactions with IgE-bound antigens that lead to cellular responses such as the degranulation of mast cells. Specifically, the FcεRIβ subunit functions as an amplifier for FcεRIγ-mediated activation signals (Lin et al, Cell 85:985-995, 1996). Thus, other structurally similar MS4A family proteins may have an important role in cellular signal transduction and they may serve as targets for therapeutic intervention. Among others, DNA and amino acid sequences of MS4A8B are reported (W09831799, WO200011150, WO0055320, WO02062946 and US20020172952). However, details of function of MS4A8B are still unclear. SUMMARY OF THE INVENTION

The present invention is based on the discovery that MS4A8B is amplified in lung tissue from COPD patients resulting in altered expression of the gene (Fig.7) relative to their expression in lung tissue of normal individuals. .

The present invention relates to novel preventive, predictive, diagnostic, prognostic and therapeutic compositions and uses for COPD. Since MS4A8B contains extracellular domains, its gene product is a particularly useful target for treatment methods as well as diagnostic and clinical monitoring methods.

The present invention further relates to novel preventive, predictive, diagnostic, prognostic and therapeutic compositions and uses for COPD based oh derivatives, fragments, analogues and homologues of the MS4A8B gene.

The present invention further relates to methods for detecting the dysregulation of MS4A8B in COPD on the DNA and mRNA levels.

The present invention further relates to a method for the prediction, diagnosis or prognosis of COPD by the detection of MS4A8B gene or MS4A8B genomic nucleic acid sequence which is altered in COPD.

In one embodiment the expression of the MS4A8B gene can be detected with arrays.

In a another embodiment the expression of the gene can be detected with beadbased direct fluorescent readout techniques such as provided by Luminex Corp. (described e.g. in US 6,268,222).

In one embodiment, the invention pertains to a method of determining the phenotype of a cell or tissue, comprising detecting the differential expression, relative to a normal or untreated cell, of the polynucleotide comprising SEQ ID NO: 1, wherein the polynucleotide is differentially expressed by at least about 1.5 fold, at least about 2 fold or at least about 3 fold.

In a further aspect the invention pertains to a method of determining the phenotype of a cell or tissue, comprising detecting the differential expression, relative to a normal or untreated cell, of at least one polynucleotide which hybridizes under stringent conditions to the polynucleotides of SEQ ID NO: 1 and encodes a polypeptide exhibiting the same biological function as MS4A8B, wherein the polynucleotide is differentially expressed by at least at least about 1.5 fold, at least about 2 fold or at least about 3 fold. In another embodiment of the invention a polynucleotide comprising a polynucleotide of SEQ ID NO: 1 or encoding the polypeptide of SEQ ID NO: 2 is used to identify cells or tissue in individuals which exhibit a phenotype predisposed to COPD or a diseased phenotype, thereby (a) predicting whether an individual is at risk for the development, or (b) diagnosing whether an individual is having, or (c) prognosing the progression or the outcome of the treatment COPD.

In yet another embodiment the invention provides a method for identifying genomic regions which are altered on the chromosomal level and encode genes that are linked by function and ate differentially expressed in COPD.

In yet another embodiment the invention provides the genomic regionl lql2, specifically the genomic region found on the human genomic sequence contig with the accession number NT_033903, for use in prediction, diagnosis and prognosis as well as prevention and treatment of COPD. In particular not only the intragenic regions, but also intergenic regions, pseudogenes or non-transcribed genes of said chromosomal regions can be used for diagnostic, predictive, prognostic and preventive and therapeutic compositions and methods.

In yet another embodiment the invention provides methods of screening for agents which regulate the activity of a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1. A test compound is contacted with a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1. Binding of the test compound to the polypeptide is detected. A test compound which binds to the polypeptide is thereby identified as a potential therapeutic agent for the treatment of COPD.

In even another embodiment the invention provides another method of screening for agents which regulate the activity of a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1. A test compound is contacted with a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1. A biological activity mediated by the polypeptide is detected. A test compound which decreases the biological activity is thereby identified as a potential therapeutic agent for decreasing the activity of the polypeptide encoded by a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 in COPD. A test compound which increases the biological activity is thereby identified as a potential therapeutic agent for increasing the activity of the polypeptide encoded by the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 in COPD.

In another embodiment the invention provides a method of screening for agents which regulate the activity of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1. A test compound is contacted with a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 . Binding of the • test compound to the polynucleotide comprising the polynucleotide of SEQ ID NO: 1 is detected. A test compound which binds to the polynucleotide is thereby identified as a potential therapeutic agent for regulating the activity of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 in COPD.

The invention thus provides polypeptides if SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 which can be used^' to identify compounds which may act, for example, as regulators or modulators such as agonists and antagonists, partial agonists, inverse agonists, activators, co-activators and inhibitors of the polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1. Accordingly, the invention provides reagents and methods for regulating a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 in COPD. The regulation can be an up- or down regulation. Reagents that modulate the expression, stability or amount of a polynucleotide comprising a polynucleotide of SEQ ID NO: 1 or the activity of the polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 can be a protein, a peptide, a peptidomimetic, a nucleic acid, a nucleic acid analogue (e.g. peptide nucleic acid, locked nucleic acid) or a small molecule. Methods that modulate the expression, stability or amount of a polynucleotide comprising a polynucleotide of SEQ ID NO: 1 or the activity of the ^" polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 can be gene replacement therapies, antisense, ribozyme, RNA interference and triplex nucleic acid approaches.

In one embodiment of the invention provides antibodies which specifically bind to a full-length or partial polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 or a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 for use in prediction, prevention, diagnosis, prognosis and treatment of COPD. Yet another embodiment of the invention is the use of a reagent which specifically binds to a polynucleotide comprising a polynucleotide of SEQ ID NO: 1 or a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 in the preparation of a medicament for the treatment of COPD.

Still another embodiment is the use of a reagent that modulates the activity or stability of a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 or the expression, amount or stability of a polynucleotide comprising a polynucleotide of SEQ ID NO: 1 in the preparation of a medicament for the treatment of COPD.

Still another embodiment of the invention is a pharmaceutical composition which includes a reagent which specifically binds to a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 or a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, and a pharmaceutically acceptable carrier.

Yet another embodiment of the invention is a pharmaceutical composition including a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 or encoding a polypeptide comprising the polypeptide of SEQ ID NO: 2.

In one embodiment, a reagent which alters the level of expression in a cell of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 or encoding a polypeptide comprising the poly- peptide of SEQ ID NO: 2, or a sequence complementary thereto, is identified by providing a cell, treating the cell with a test reagent, determining the level of expression in the cell of a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 or encoding a polypeptide comprising the polypeptide of SEQ ID NO: 2 or a sequence complementary thereto, and comparing the level of expression of the polynucleotide in the treated cell with the level of expression of the poly- nucleotide in an untreated cell, wherein a change in the level of expression of the polynucleotide in the treated cell relative to the level of expression of the polynucleotide in the untreated cell is indicative of an agent which alters the level of expression of the polynucleotide in a cell.

The invention further provides a pharmaceutical composition comprising a reagent identified by this method.

Another embodiment of the invention is a pharmaceutical composition which includes a polypeptide comprising the polypeptide of SEQ ID NO: 2 or which is encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1. A further embodiment of the invention is a pharmaceutical composition comprising a polynucleotide including a sequence which hybridizes under stringent conditions to a polynucleotide comprising a polynucleotide of SEQ ID NO: 1 and encoding a polypeptide exhibiting the same biological function as MS4A8B, or encoding a polypeptide of SEQ ID NO: 2 . Pharmaceutical compositions, useful in the present invention may further include fusion proteins comprising a polypeptide comprising a polynucleotide of SEQ ID NO: 1, or a fragment thereof, antibodies, or antibody fragments

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the DNA sequence encoding a human MS4A8B polypeptide.

Fig. 2 shows the amino acid sequence deduced from the DNA sequence of Fig. 1.

Fig. 3 shows PCR. primers used to amplify DNA complementary to human MS4A8B mRNAs in various tissues.

Fig. 4 shows a PCR primer used to amplify DNA complementary to human MS4A8B mRNA in various tissues.

Fig. 5 shows the probe used 'to detect the expression of human MS4A8B mRNA in various tissues.

Fig. 6 shows the probe used to detect the expression of human MS4A8B mRNA in various tissues.

Fig. 7 shows the relative expression levels for human ^' MS4A8B obtained from microarray experiments using various lung samples.

Fig. 8 shows the expression profiles of human MS4A8B in various lung samples.

Fig. 9 shows the expression profiles of human MS4A8B in various tissues.

Fig. 10 shows the expression profiles of human MS4A8B in various cells. DETAIXED DESCRIPTION OF THE INVENTION

DEFINITIONS

"Differential expression", as used herein, refers to both quantitative as well as qualitative differences in the genes' expression patterns depending on differential development arid/or reaction to lipid environment of macrophages. Differentially expressed genes may represent "marker genes," and/or "target genes". The expression pattern of a differentially expressed gene disclosed herein may be utilized as part of a prognostic or diagnostic COPD evaluation., Alternatively, a differentially expressed gene disclosed herein may be used in methods for identifying reagents and compounds and uses of these reagents and compounds for the treatment of COPD as well as methods of treatment.

"Biological activity" or "bioactivity" or "activity" or "biological function", which are used interchangeably, herein mean an effector or antigenic function that is directly or indirectly performed by a polypeptide of MS4A8B, or by any fragment thereof in vivo or in vitro. Biological activities include but are not limited to binding to polypeptides, binding to other proteins or molecules, enzymatic activity, signal transduction, activity as a DNA binding protein, as a transcription regulator, ability to bind damaged DNA, etc. A bioactivity can be modulated by directly affecting the subject polypeptide. Alternatively, a bioactivity can be altered by modulating the level of the polypeptide, such as by modulating expression of the corresponding gene.

The term "marker" or "biomarker" refers a biological molecule, e.g., a nucleic acid, peptide, hormone, etc., whose presence or concentration can be detected and correlated with a known condition, such as a disease state.

The term "biological sample", as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a "clinical sample" which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, cell-containing bodyfluids, free floating nucleic acids, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

By "array" or "matrix" is meant an arrangement of addressable locations or "addresses" on a device. The locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats. The number of locations can range from several to at least hundreds of thousands. Most importantly, each location represents a totally independent reaction site. Arrays include but are not limited to nucleic acid arrays, protein arrays and antibody arrays. A "nucleic acid array" refers to an array containing nucleic acid probes, such as oligonucleotides, polynucleotides or larger portions of genes. The nucleic acid on the array is preferably single stranded. Arrays wherein the probes are oligonucleotides are referred to as "oligonucleotide arrays" or "oligonucleotide chips." A "microarray," herein also refers to a "biochip" or "biological chip", an array of regions having a density of discrete regions of at least about 100/cm², and preferably at least about 1000/cm². The regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 μm, and are separated from other regions in the array by about the same distance. A "protein array" refers to an array containing polypeptide probes or protein probes which can be in native form or denatured. An "antibody array" refers to an array containing antibodies which include but are not limited to monoclonal antibodies (e.g. from a mouse), chimeric antibodies, humanized antibodies or phage antibodies and single chain antibodies as well as fragments from antibodies.

The term "agonist", as used herein, is meant to refer to an agent that mimics or upregulates (e.g., potentiates or supplements) the bioactivity of a protein. An agonist can be a wild-type protein or derivative thereof having at least one bioactivity of the wild-type protein. An agonist can also be a compound that upregulates expression of a gene or which increases at least one bioactivity of a protein. An agonist can also be a compound which increases the interaction of a polypeptide with another molecule, e.g., a target peptide or nucleic acid.

The term "antagonist" as used herein is meant to refer to an agent that downregulates (e.g., suppresses or inhibits) at least one bioactivity of a protein. An antagonist can be a compound which inhibits or decreases the interaction between a protein and another molecule, e.g., a target peptide, a ligand or an enzyme substrate. An antagonist can also be a compound that downregulates expression of a gene or which reduces the amount of expressed protein present.

"Small molecule" as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides,^' peptidomimetics, carbohydrates, lipids or other organic (carbon- containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate a bioactivity.

The terms "modulated" or "modulation" or "regulated" or "regulation" and "differentially regulated" as used herein refer to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating) and down regulation [i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)].

"Transcriptional regulatory unit" refers to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription, of protein coding sequences with which they are operably linked. In preferred embodiments, transcription ofone of the genes is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally occurring forms of the polypeptide.

The term "derivative" refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

The term "nucleotide analog" refers to oligomers or polymers being at least in one feature different from naturally occurring nucleotides, oligonucleotides or polynucleotides, but exhibiting functional features of the respective naturally occurring nucleotides (e.g. base paring, hybridization, coding information) and that can be used for said compositions. The nucleotide analogs can consist of non-naturally occurring bases or polymer backbones, examples of which are LNAs, PNAs and Morpholinos. The nucleotide analog has at least one molecule different from its naturally occurring counterpart or equivalent.

"MS4A8B GENE" or "MS4A8B GENE" as used herein refers to the polynucleotides of SEQ ID NO: 1, as well as derivatives, fragments, analogs and homologues thereof, the polypeptides encoded thereby, the polypeptide of SEQ ID NO: 2 as well as derivatives, fragments, analogs and homologues thereof and the corresponding genomic transcription units which can be derived or identified with standard techniques well known in the art.

The term "chromosomal region" as used herein refers to a consecutive DNA stretch on a chromosome which can be defined by cytogenetic or other genetic markers such as e.g. restriction length polymorphisms (RFLPs), single nucleotide polymorphisms (SNPs), expressed sequence tags (ESTs), sequence tagged sites (STSs), microsatellites and genes. Typically a chromosomal region consists of up to 2 Megabases (MB), up to 4 MB, up to 6 MB, up to 8 MB, up to 10 MB, up to 20 MB or even more MB.

The term "altered chromosomal region" or" aberrant chromosomal region" refers to a structural change of the chromosomal composition and DNA sequence, which can occur by the following events: amplifications, deletions, inversions, insertions, translocations and or viral integrations/ A trisomy, where a given cell harbors more than two copies of a chromosome, is within the meaning of the term "amplification" of a chromosome or chromosomal region.

The present invention provides polynucleotide sequences and proteins encoded thereby, as well as probes derived from the polynucleotide sequences, antibodies directed to the encoded proteins, and predictive, preventive, diagnostic, prognostic and therapeutic uses for individuals which are at risk for or which have COPD.

It is a discovery of the present invention that human MS4A8B can be regulated to control diseases that are caused by aberrant activity of this polypeptide and diseases whose symptoms can be ameliorated by stimulating or inhibiting the activity of MS4A8B.

The differential gene expression between lung tissues obtained from two healthy individuals and two patients diagnosed with chronic obstructive pulmonary disease (COPD) is detected. Among the genes showing higher expression in COPD compared to healthy lung, MS4A8B is found as a gene after further confirmation study by quantitative PCR as described.

Present inventors' findings that the expression of MS4A8B is dominant in human normal lung and trachea, and the high expression of MS4A8B was observed in lung tissue from COPD patients compared to normal counterparts. These data support the idea that MS4A8B may have an important role in the pathogenesis of COPD. Therefore, the modulation of MS4A8B may be useful approach to provide an effective and selective therapy on diseases caused by respiratory tissues oriented diseases, such as COPD.

Identification of differential expression

Transcripts within the collected RNA samples which represent RNA produced by differentially expressed genes may be identified by utilizing a variety of methods which are known to those of skill in the art. For example, differential screening [Tedder, T. F. et al., 1988, (79)], subtractive hybridization [Hedrick, S. M. et al., 1984, (80); Lee, S. . et al., 1984, (81)], and, preferably, differential display (Liang, P., and Pardee, A. B., 1993, U.S. Pat. No. 5,262,311, which is incorporated herein by reference in its entirety), may be utilized to identify polynucleotide sequences derived from genes that are differentially expressed.

Differential screening involves the duplicate screening of a cDNA library in which one copy of the library is screened with a total cell cDNA probe corresponding to the mRNA population of one cell type while a duplicate copy of the cDNA library is screened with a total cDNA probe corresponding to the mRNA population of a second cell type. For example, one cDNA probe may correspond to a total cell cDNA probe of a cell type derived from a control subject, while the second cDNA probe may correspond to a total cell cDNA probe of the same cell type derived from an experimental subject. Those clones which hybridize to one probe but not to the other potentially represent clones derived from genes differentially expressed in the cell type of interest in control versus experimental subjects.

Subtractive hybridization techniques generally involve the isolation of mRNA taken from two different sources, e.g., control and experimental tissue, the hybridization of the mRNA or single- stranded cDNA reverse-transcribed from the isolated mRNA, and the removal of all hybridized, and therefore double-stranded, sequences. The remaining non-hybridized, single-stranded cDNAs, potentially represent clones derived from genes that are differentially expressed in the two mRNA sources. Such single-stranded cDNAs are then used as the starting material for the construction of a library comprising clones derived from differentially expressed genes.

The differential display technique describes a procedure, utilizing the well known polymerase chain reaction (PCR; the experimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat. No.

4,683,202) which allows for the identification of sequences derived from genes which are differentially expressed. First, isolated RNA is reverse-transcribed into single-stranded cDNA, utilizing standard techniques which are well known to those of skill in the art. Primers for the reverse transcriptase reaction may include, but are not limited to, oligo dT-containing primers, preferably of the reverse primer type of oligonucleotide described below. Next, this technique uses pairs of PCR primers, as described below, which allow for the amplification of clones representing a random subset of the RNA transcripts present within any given cell. Utilizing different pairs of primers allows each of the mRNA transcripts present in a cell to be amplified. Among such amplified transcripts may be identified those which have been produced from differentially ■ expressed genes.

The reverse oligonucleotide primer of the primer pairs may contain an oligo dT stretch of nucleotides, preferably eleven nucleotides long, at its 5' end, which hybridizes to the poly(A) tail of mRNA or to the complement of a cDNA reverse transcribed from an mRNA poly(A) tail. Second, in order to increase the specificity of the reverse primer, the primer may contain one or more, preferably two, additional nucleotides at its 3' end. Because, statistically, only a subset of the mRNA derived sequences present in the sample of interest will hybridize to such primers, the additional nucleotides allow the primers to amplify only a subset of the mRNA derived sequences present in the sample of interest. This is preferred in that it allows more accurate and complete visualization and characterization of each of the bands representing amplified sequences.

The forward primer may contain a nucleotide sequence expected, statistically, to have the ability to hybridize to cDNA sequences derived from the tissues of interest. The nucleotide sequence may be an arbitrary one, and the length of the forward oligonucleotide primer may range from about 9 to about 13 nucleotides, with about 10 nucleotides being preferred. Arbitrary primer sequences cause the lengths of the amplified partial cDNAs produced to be variable, thus allowing different clones to be separated by using standard denaturing sequencing gel electrophoresis. PCR reaction conditions should be chosen which optimize amplified product yield and specificity, and, additionally, produce amplified products of lengths which may be resolved utilizing standard gel electrophoresis techniques. Such reaction conditions are well known to those of skill in the art, and important reaction parameters include, for example, length and nucleotide sequence of oligonucleotide primers as discussed above, and annealing and elongation step temperatures and reaction times. The pattern of clones resulting from the reverse transcription and amplification of the mRNA of two different cell types is displayed via sequencing gel electrophoresis and compared. Differences in the two banding patterns indicate potentially differentially expressed genes.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' nontranscribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer; ABI), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

Once potentially differentially expressed gene sequences have been identified via bulk techniques such as, for example, those described above, the differential expression of such putatively differentially expressed genes should be corroborated. Corroboration may be accomplished via, for example, such well known techniques as Northern analysis and/or RT-PCR. Upon corroboration, the differentially expressed genes may be further characterized, and may be identified as target, and/or marker genes, as discussed, below.

Also, amplified sequences of differentially expressed genes obtained through, for example, differential display may be used to isolate full length clones of the corresponding gene. The full length coding portion of the gene may readily be isolated, without undue experimentation, by molecular biological techniques well known in the art. For example, the isolated differentially expressed amplified fragment may be labeled and used to screen a cDNA library. Alternatively, the labeled fragment may be used to screen a genomic library.

An analysis of the tissue distribution of the mRNA produced by the identified genes may be conducted, utilizing standard techniques well known to those of skill in the art. Such techniques may include, for example, Northern analyses and RT-PCR. Such analyses provide information as to whether the identified genes are expressed in tissues expected to contribute to COPD. Such " analyses may also provide quantitative information regarding steady state mRNA regulation, yielding data concerning which of the identified genes exhibits a high level of regulation in, preferably, tissues which may be expected to contribute to COPD.

Such analyses may also be performed on an isolated cell population of a particular cell type derived from a given tissue. Additionally, standard in situ hybridization techniques may be utilized to provide information regarding which cells within a given tissue express the identified gene. Such analyses may provide information regarding the biological function of an identified gene relative to COPD in instances wherein only a subset of the cells within the tissue is thought to be relevant to COPD.

Polypeptides

MS4A8B polypeptides according to the invention comprise at least 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, or 250 contiguous amino acids from the amino acid sequence shown in SEQ ID NO. 2 or a biologically active variant thereof, as defined below. An MS4A8B polypeptide of the invention therefore can be a portion of an MS4A8B, a full-length MS4A8B, or a fusion protein comprising all or a portion of an MS4A8B.

Biologically Active Variants

MS4A8B polypeptide variants which are biologically active, i.e., retain the ability to bind a ligand 5 to produce a biological effect, such as mobilization of intracellular calcium, or phosphoinositide metabolism, also are MS4A8B polypeptides. Preferably, naturally or non-naturally occurring MS4A8B polypeptide variants have amino acid sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to an amino acid sequence shown in SEQ ID NO. 2 or a fragment thereof. Percent identity between a putative MS4A8B polypeptide variant and an amino 0 acid sequence of SEQ ID NO. 2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes) .Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are 5 substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine ' ^"' with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological 0 activity of an MS4A8B polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active MS4A8B polypeptide can readily be determined by assaying for binding to a ligand or by conducting a functional assay, such as those described in the specific examples, below.

Fusion Proteins

5 Fusion proteins are useful for generating antibodies against MS4A8B polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of an MS4A8B polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two- hybrid or phage display systems, can be used for this purpose. Such methods are well known in 0 . the art and also can be used as drug screens.

An MS4A8B polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, or 250 or more contiguous amino acids of SEQ ID NO. 2 or a biologically active variant thereof. The first polypeptide segment also can comprise full-length MS4A8B.

The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetylt ansferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the MS4A8B polypeptide-encoding sequence and the heterologous protein sequence, so that the MS4A8B polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO. 1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Polynucleotides

An MS4A8B polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for an MS4A8B polypeptide. A nucleotide sequence encoding the MS4A8B having SEQ ID NO. 2 is shown in SEQ ID NO. 1.

Degenerate nucleotide sequences encoding human MS4A8B polypeptides, as well as homologous nucleotide sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence shown in SEQ ID NO. 1 or its complement also are MS4A8B polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of MS4A8B polynucleotides which encode biologically active MS4A8B polypeptides also are MS4A8B polynucleotides.

Identification of Polynucleotide Variants and Homoloss

Variants and homologs of the MS4A8B polynucleotides described above also are MS4A8B polynucleotides. Typically, homologous MS4A8B polynucleotide sequences can be identified by hybridization of candidate polynucleotides. to known MS4A8B polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice,^' 30 minutes each; then 2X SSC, 0.1% SDS, 50°C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the MS4A8B polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of MS4A8B polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_m of a double-stranded DNA decreases by 1-1.5°C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, Wi (1973). Variants of human MS4A8B polynucleotides or MS4A8B polynucleotides of other species can therefore be identified by hybridizing a putative homologous MS4A8B polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO. 1 or 3 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to MS4A8B polynucleotides or their complements following stringent hybridization and/or wash conditions also are MS4A8B polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51. Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20°C below the calculated T_m of the hybrid under study. The T_m of a hybrid between an MS4A8B polynucleotide having a nucleotide sequence shown in SEQ ID NO. 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 4% 1390 (1962):

T_m = 81.5°C - 16.6(log₁₀[Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 600//), where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide, 4X SSC at 42°C, or 0.5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.

Preparation of Polynucleotides

An MS4A8B polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated MS4A8B polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises MS4A8B nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.

MS4A8B cDNA molecules can be made with standard molecular biology techniques, using MS4A8B mRNA as a template. MS4A8B cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used . to synthesizes MS4A8B polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode an MS4A8B polypeptide having, for example, an amino acid sequence shown in SEQ ID NO. 2 or a biologically active variant thereof. Extendins Polynucleotides

Various PCR-based methods can be used to extend the nucleic acid sequences encoding human MS4A8B to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50%) or more, and to anneal to the target sequence at temperatures about 68-72°C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions. Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four^' different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

Obtaining Polypeptides

MS4A8B polypeptides can be obtained, for example, by purification from human cells, by expression of MS4A8B polynucleotides, or by direct chemical synthesis.

Protein Purification

MS4A8B polypeptides can be purified from any cell which expresses the receptor, including host cells which have been transfected with MS4A8B polynucleotides which express such polypeptides. Colon, adenocarcinoma, liver, and kidney are particularly useful sources of HM74-like polypeptides. A purified MS4A8B polypeptide is separated from other compounds which normally associate with the MS4A8B polypeptide in the cell, such as certain proteins, carbohy- drates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, . ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

An MS4A8B polypeptide can be conveniently isolated as a complex with its associated protein, as described in the specific examples, below. A preparation of purified MS4A8B polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.

Expression of Polynucleotides

To express an MS4A8B polypeptide, an MS4A8B polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding MS4A8B polypeptides and appropriate transcriptional and franslational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding an MS4A8B polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast fransformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g. , baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions ~ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.

Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the

■ BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding an MS4A8B polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the MS4A8B polypeptide. For example, when a large quantity of an MS4A8B polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the MS4A8B polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β- galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol. 153, 516-544, 1987.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding MS4A8B polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671- 1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated fransfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express an MS4A8B polypeptide. For example, in one such '^' system Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichophisia larvae. Sequences encoding MS4A8B polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under confrol of the polyhedrin promoter. Successful insertion of MS4A8B polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which MS4A8B polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994). Mammalian Expression Systems

A number of viral-based expression systems can be used to express MS4A8B polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding MS4A8B polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing an MS4A8B polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding MS4A8B polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding an MS4A8B polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational confrol signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Differ. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed MS4A8B polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express MS4A8B polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on^' the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced MS4A8B sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.

Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosylfransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tk^" or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the amino- glycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetylfransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its subsfrate GUS, and luciferase and its subsfrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).

Detecting Expression of Polypeptides

Although the presence of marker gene expression suggests that the MS4A8B polynucleotide is. also present, its presence and expression may need to be confirmed. For example, if a sequence encoding an MS4A8B polypeptide is inserted within a marker gene sequence, fransformed cells containing sequences which encode an MS4A8B polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding an MS4A8B polypeptide under the confrol of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the MS4A8B polynucleotide.

Alternatively, host cells which contain an MS4A8B polynucleotide and which express an MS4A8B polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding an MS4A8B polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding an MS4A8B polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding an MS4A8B polypeptide to detect transformants which contain an MS4A8B polynucleotide.

A variety of protocols for detecting and measuring the expression of an MS4A8B polypeptide,

^' using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art.

Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on an MS4A8B polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et α/., J Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding MS4A8B polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding an MS4A8B polypeptide can be cloned into a vector for the production of a mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding an MS4A8B polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a fransformed cell can be secreted or contained intracellularly . depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode MS4A8B polypeptides can be designed to contain signal sequences which direct secretion of soluble MS4A8B polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound MS4A8B polypeptide.

As discussed above, other constructions can be used to join a sequence encoding an MS4A8B polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the MS4A8B polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing an MS4A8B polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage, site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al, Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the MS4A8B polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.

Chemical Synthesis

• Sequences encoding an MS4A8B polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, an MS4A8B polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of MS4A8B polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic MS4A8B polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the MS4A8B polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce MS4A8B polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can .be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter MS4A8B polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Predictive, Diagnostic and Proφτostic Assays

The present invention provides method for determining whether a subject is at risk for developing COPD by detecting the disclosed biomarkers, i.e., the disclosed polynucleotide marker comprising the polynucleotide sequence of the SEQ ID NO: 1 and/or the polypeptide markers encoded thereby or polypeptide markers comprising the polypeptide sequences of the SEQ ID NO: 2 for COPD.

In clinical applications, biological samples can be screened for the presence and/or absence of the biomarkers identified herein. Such samples are for example needle biopsy cores, surgical resection samples, or body fluids like serum, thin needle nipple aspirates and urine. For example, these methods include obtaining a biopsy, which is optionally fractionated by cryostat sectioning to enrich diseases cells to about 80% of the total cell population. In certain embodiments, polynucleotides extracted from these samples may be amplified using techniques well known in the art. The expression levels of selected markers detected would be compared with statistically valid groups of diseased and healthy samples.

In one embodiment the diagnostic method comprises determining whether a subject has an abnormal mRNA and/or protein level of the disclosed markers, such as by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immuno- precipitation, Western blot hybridization, or immunohistochemistry. According to the method, cells are obtained from a subject and the levels of the disclosed biomarkers, protein or mRNA level, is determined and compared to the level of these markers in a healthy subject. An abnormal level of the biomarker polypeptide or mRNA levels is likely to be indicative of COPD such as COPD.

In another embodiment the diagnostic method comprises determining whether a subject has an abnormal DNA content of said genes or said genomic loci, such as by Southern blot analysis, dot blot analysis, Fluorescence or Colorimetric In Situ Hybridization, Comparative Genomic Hybridization or quantitative PCR. In general these assays comprise the usage of probes from representative genomic regions.. The probes contain at least parts of said genomic regions or sequences complementary or analogous to said regions. In particular infra- or intergenic regions of said genes or genomic regions. The probes can consist of nucleotide sequences or sequences of analogous functions (e.g. PNAs, Morpholino oligomers) being able to bind to target regions by hybridization. In general genomic regions being altered in said patient samples are compared with unaffected control samples (normal tissue from the same or different patients, surrounding unaffected tissue, peripheral blood) or with genomic regions of the same sample that don't have said alterations and can therefore serve as internal controls. In a preferred embodiment regions located on the same chromosome are used. Alternatively, gonosomal regions and /or regions with defined varying amount in the sample are used. In one favored embodiment the DNA content, structure, composition or modification is compared that lie within distinct genomic regions. Especially favored are methods that detect the DNA content of said samples, where the amount of target regions are altered by amplification and or deletions. In another embodiment the target regions are analyzed for the presence of polymorphisms (e.g. Single Nucleotide Polymorphisms or mutations) that affect or predispose the cells in said samples with regard to clinical aspects, being of diagnostic, prognostic or therapeutic value. Preferably, the identification of sequence variations is used to define haplotypes that result in characteristic behavior of said samples with said^' clinical aspects.

One embodiment of the invention is a method for the prediction, diagnosis or prognosis of COPD by the detection of at least 10, at least 5, or at least 4, or at least 3 and more preferably at least 2 markers whereby the markers are genes and/or genomic nucleic acid sequences that are located on one chromosomal region which is altered in COPD.

One further embodiment of the invention is a method for the prediction, diagnosis or prognosis of COPD by the detection of MS4A8B gene and/or genomic nucleic acid sequence.

In one embodiment, the method for the prediction, diagnosis or prognosis of COPD and COPD in particular is done by the detection of:

(a) polynucleotide of the SEQ ID NO: 1 ;

(b) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) encoding a polypeptide exhibiting the same biological function as MS4A8B;

(c) a polynucleotide the sequence of which deviates from the polynucleotide specified in (a) ^' and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B;

(d) a polynucleotide which represents a specific fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (c) encoding a polypeptide exhibiting the same biological function as MS4A8B;

in a biological sample comprising the following steps: hybridizing any polynucleotide or analogous oligomer specified in (a) to (d) to a polynucleotide material of a biological sample, thereby forming a hybridization complex; and detecting said hybridization complex.

In another embodiment the method for the prediction, diagnosis or prognosis of COPD is done as just described but, wherein before hybridization, the polynucleotide material of the biological sample is amplified.

In another embodiment the method for the diagnosis or prognosis of COPD in particular is done by the detection of:

(a) a polynucleotide selected from the polynucleotides of the SEQ ID NO: 1; (b) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) encoding a polypeptide exhibiting the same biological function as MS4A8B;

(c) a polynucleotide the sequence of which deviates from the polynucleotide specified in (a) and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B;

(e) a polypeptide encoded by a polynucleotide sequence specified in (a) to (d)

(f) a polypeptide comprising the polypeptide of SEQ ID NO: 2:

comprising the steps of contacting a biological sample with a reagent which specifically interacts with the polynucleotide specified in (a) to (d) or the polypeptide specified in (e).

L DNA array technology

In one embodiment, the present Invention also provides a method wherein polynucleotide probes are immobilized an a DNA chip in an organized array. Oligonucleotides can be bound to a solid Support by a variety of processes, including lithography. For example a chip can hold up to 41,000 oligonucleotides (GeneChip, Affymetrix). The present invention provides significant advantages over the available tests for COPD, such as COPD, because it increases the reliability of the test by providing an array of polynucleotide markers on a single chip.

The method includes obtaining a biopsy of an^* affected person, which is optionally fractionated by cryostat sectioning to enrich diseased cells to about 80% of the total cell population and the use of body fluids such as serum or urine, serum or cell containing liquids (e.g. derived from fine needle aspirates). The DNA or RNA is then extracted, amplified, and analyzed with a DNA chip to determine the presence of absence of the marker polynucleotide sequences. In one embodiment, the polynucleotide probes are spotted onto a subsfrate in a two-dimensional matrix or array, samples of polynucleotides can be labeled and then hybridized to the probes. Double-stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away.

The probe polynucleotides can be spotted on substrates including glass, nitrocellulose, etc. The probes can be bound to the Substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. The sample polynucleotides can be labeled using radioactive labels, fluorophores, chromophores, etc. Techniques for constructing arrays and methods of using these arrays are described in EP No. 0 799 897; PCT No. WO 97/29212; PCT No. WO 97/27317; EP No. 0 785 280; PCT No. WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP No. 0 728 520; U.S. Pat. No. 5,599,695; EP No. 0 721 016; U.S. Pat. No. 5,556,752; PCT No. WO 95/22058; and U.S. Pat. No. 5,631,734. Further, arrays can be used to examine differential expression of genes and can be used to determine gene function. For example, arrays of the instant polynucleotide sequences can be used to determine if any of the polynucleotide sequences are differentially expressed between normal cells and diseased cells, for example. High expression of a particular message in a diseased sample, which is not observed in a corresponding normal sample, can indicate a COPD specific protein.

Accordingly, in one aspect, the invention provides probes and primers that are specific to the unique polynucleotide markers disclosed herein.

In one embodiment, the method comprises using a polynucleotide probe to determine the presence of malignant or COPD cells in particular in a tissue from a patient. Specifically, the method comprises:

1) providing a polynucleotide probe comprising a nucleotide sequence at least 12 nucleotides in length, preferably at least 15 nucleotides, more preferably, 25 nucleotides, and most preferably at least 40 nucleotides, and up to all or nearly all of the coding sequence which , is complementary to a portion of the coding sequence of a polynucleotide of the SEQ ID

NO: 1 or a sequence complementary thereto and is

2) differentially expressed in COPD;

3) obtaining a tissue sample from a patient with COPD;

4) providing a second tissue sample from a patient with no COPD;

5) contacting the polynucleotide probe under stringent conditions with RNA of each of said first and second tissue samples (e.g., in a Northern blot or in situ hybridization assay); and

6) comparing (a) the amount of hybridization of the probe with RNA of the first tissue sample, with (b) the amount of hybridization of the probe with RNA of the second tissue sample; wherein a statistically significant difference in the amount of hybridization with the RNA of the first tissue sample as compared to the amount of hybridization with the RNA of the second tissue sample is indicative of COPD and COPD in particular in the first tissue sample.

2. Data analysis methods

Comparison of the expression levels of one or more "MS4A8B GENE" with reference expression levels, e.g., expression levels in diseased cells of COPD or in normal counterpart cells, is preferably conducted using computer systems. In one embodiment, expression levels are obtained in two cells and these two sets of expression levels are introduced into a computer system for comparison. In a preferred embodiment, one set of expression levels is entered into a computer system for comparison with values that are already present in the computer system, or in computer-readable form that is then entered into the computer system.

In one embodiment, the invention provides a computer readable form of the gene expression profile data of the invention, or of values corresponding to the level of expression of at least one "MS4A8B GENE" in a diseased cell. The values can be mRNA expression levels obtained from experiments, e.g., microarray analysis. The values can also be mRNA levels normalized relative to a reference gene whose expression is constant in numerous cells under numerous conditions, e.g., GAPDH. In other embodiments, the values in the computer are ratios of, or differences between, normalized or non-normalized mRNA levels in different samples.

The gene expression profile data can be in the form of a table, such as an Excel table. The data can be alone, or it can be part of a larger database, e.g., comprising other expression profiles. For example, the expression profile data of the invention can be part of a public database. The computer readable form can be in a computer. In another embodiment, the invention provides a computer displaying the gene expression profile data.

In one embodiment, the invention provides a method for determining the similarity between the level of expression of one or more "MS4A8B GENE" in a first cell, e.g., a cell of a subject, and that in a second cell, comprising obtaining the level of expression of one or more "MS4A8B GENE" in a first cell and entering these values into a computer comprising a database including records comprising values corresponding to levels of expression of one or more "MS4A8B GENE" in a second cell, and processor instructions, e.g., a user interface, capable of receiving a selection of one or more values for comparison purposes with data that is stored in the computer. The computer may further comprise a means for converting the comparison data into a diagram or chart or other type of output. In another embodiment, values representing expression levels of "MS4A8B GENE" are entered into a computer system, comprising one or more databases with reference expression levels obtained from more than one cell. For example, the computer comprises expression data of diseased and normal cells. Instructions are provided to the computer, and the computer is capable of comparing the data entered with the data in the computer to determine whether the data entered is more similar to that of a normal cell or of a diseased cell.

In another embodiment, the computer comprises values of expression levels in cells of subjects at different stages of COPD, and the computer is capable of comparing expression data entered into the computer with the data stored, and produce results indicating to which of the expression profiles in the computer,. the one entered is most similar, such as to determine the stage of COPD in the subject.

In yet another embodiment, the reference expression profiles in the computer are expression profiles from cells of COPD of one or more subjects, which cells are treated in vivo or in vitro with a drug used for therapy of COPD. Upon entering of expression data of a cell of a subject treated in vitro or in vivo with the drug, the computer is instructed to compare the data entered to the data in the computer, and to provide results indicating whether the expression data input into the computer are more similar to those of a cell of a subject that is responsive to the drug or more similar to those of a cell of a subject that is not responsive to the drug. Thus, the results indicate whether the subject is likely to respond to the freatment with the drug or unlikely to respond to it.

In one embodiment, the invention provides a^' system that comprises a means for receiving gene expression data for one or a plurality of genes; a means for comparing the gene expression data from each of said one or plurality of genes to a common reference frame; and a means for presenting the results of the comparison. This system may further comprise a means for clustering the data.

In another embodiment, the invention provides a computer program for analyzing gene expression data comprising (i) a computer code that receives as input gene expression data for a plurality of genes and (ii) a computer code that compares said gene expression data from each of said plurality of genes to a common reference frame.

The invention also provides a machine-readable or computer-readable medium including program instructions for performing the following steps: (i) comparing a plurality of values corresponding to expression levels of one or more genes characteristic of COPD in a query cell with a database including records comprising reference expression or expression profile data of one or more reference cells and an annotation of the type of cell; and (ii) indicating to which cell the query cell is most similar based on similarities of expression profiles. The reference cells can be cells from subjects at different stages of COPD. The reference cells can also be cells from subjects responding or not responding to a particular drug treatment and optionally incubated in vitro or in vivo with the drug.

The reference cells may also be cells from subjects responding or not responding to several different treatments, and the computer system indicates a preferred treatment for the subject. Accordingly, the invention provides a method for selecting a therapy for a patient having COPD, the method comprising: (i) providing the level of expression of one or more genes characteristic of - COPD in a diseased cell of the patient; (ii) providing a plurality of reference profiles, each associated with a therapy, wherein the subject expression profile and each reference profile has a plurality of values, each value representing the level of expression of a gene characteristic of COPD; and (iii) selecting the reference profile most similar to the subject expression profile, to thereby select a therapy for said patient. In a preferred embodiment step (iii) is performed by a computer. The most similar reference profile may be selected by weighing a comparison value of the plurality using a weight value associated with the corresponding expression data.

The relative abundance of an mRNA in two biological samples can be scored as a perturbation and its magnitude determined (i.e., the abundance is different in the two sources of mRNA tested), or as not perturbed (i.e., the relative abundance is the same). In various embodiments, a difference between the two sources of RNA of at least a factor of about 25% (RNA from one source is 25% more abundant in one source than the other source), more usually about 50%, even more often by a factor of about 2 (twice as abundant), 3 (three times as abundant) or 5 (five times as abundant) is scored as a perturbation. Perturbations can be used by a computer for calculating and expression comparisons.

Preferably, in addition to identifying a perturbation as positive or negative, it is advantageous to determine the magnitude of the perturbation. This can be carried out, as noted above, by calculating the ratio of the emission of the two fluorophores used for differential labeling, or by analogous methods that will be readily apparent to those of skill in the art.

The computer readable medium may further comprise a pointer to a descriptor of a stage of COPD or to a treatment for COPD.

In operation, the means for receiving gene expression data, the means for comparing the gene expression data, the means for presenting, the means for normalizing, and the means for clustering within the context of the systems of the present invention can involve a programmed computer with the respective. functionalities described herein, implemented in hardware or hardware and software; a logic circuit or other component of a programmed computer that performs the operations specifically identified herein, dictated by a computer program; or a computer memory encoded with executable instructions representing a computer program that can cause a computer to function in the particular fashion described herein.

Those skilled in the art will understand that the systems and methods of the present invention may be applied to a variety of systems, including IBM-compatible personal computers running MS- DOS or Microsoft Windows.

The computer may , have internal components linked to external components. The internal components may include a processor element interconnected with a main memory. The computer system can be an Intel Pentium^®-based processor of 200 MHz or greater clock rate and with 32 MB or more of main memory. The external component may comprise a mass storage, which can be one or more hard disks (which are typically packaged together with the processor and memory). Such hard disks are typically of 1 GB or greater storage capacity. Other external components include a user interface device, which can be a monitor, together with an inputting device, which can be a "mouse", or other graphic input devices, and/or a keyboard. A printing device can also be attached to the computer.

Typically, the computer system is also linked to a network link, which can be part of an Ethernet link to other local computer systems, remote computer systems, or wide area communication networks, such as the Internet. This network link allows the computer system to share data and processing tasks with other computer systems.

Loaded into memory during operation of this system are several software components, which are both standard in the art and special to the instant invention. These software components collectively cause the computer system to function according to the methods of this invention. These software components are typically stored on a mass storage. A software component represents the operating system, which is responsible for managing the computer system and its network interconnections. This operating system can be, for example, of the Microsoft Windows' family, such as Windows 95, Windows 98, or Windows NT. A software component represents common languages and functions conveniently present on this system to assist programs implementing the methods specific to this invention. Many high or low level computer languages can be used to program the analytic methods of this invention. Instructions can be interpreted during run-time or compiled. Preferred languages include C/C++, and JAVA^®. Most preferably, the methods of this invention are programmed in mathematical software packages which allow symbolic entry of equations and high-level specification of processing, including algorithms to be used, thereby freeing a user of the need to procedurally program individual equations or. algorithms. Such packages include Matlab from Mathworks (Natick, Mass.), Mathematica from Wolfram Research (Champaign, 111.), or S-Plus from Math Soft (Cambridge, Mass.). Accordingly, a software component represents the analytic methods of this invention as programmed in a procedural language or symbolic package. In a preferred embodiment, the computer system also contains a database comprising values representing levels of expression of one or more genes characteristic of COPD. The database may contain one or more expression profiles of genes characteristic of COPD in different cells.

In an exemplary implementation, to practice the methods of the present invention, a user first loads expression profile data into the computer system. These data can be directly- entered by the user from a monitor and keyboard, or from other computer systems linked by a network connection, or on removable storage media such as a CD-ROM or floppy disk or through the network. Next the user causes execution of expression profile analysis software which performs the steps of comparing and, e.g., clustering co-varying genes into groups of genes.

In another exemplary implementation, expression profiles are compared using a method described in U.S. Patent No. 6,203,987. A user first loads expression profile data into the computer system. Geneset profile definitions are loaded into the memory from the storage media or from a remote computer, preferably from a dynamic geneset database system, through the network. Next the user causes execution of projection software which performs the steps of converting expression profile to projected expression profiles. The projected expression profiles are then displayed.

In yet another exemplary implementation, a user first leads a projected profile into the memory. The user then causes the loading of a reference profile into the memory. Next, the user causes the execution of comparison software which performs the steps of objectively comparing the profiles.

3. Detection of variant polynucleotide sequence

In yet another embodiment, the invention provides methods for determining whether a subject is at risk for developing a disease, such as a predisposition to develop COPD, for example COPD, associated with an aberrant activity of any one of the polypeptides encoded by any of the polynucleotides of the SEQ ID NO: 1, wherein the aberrant activity of the polypeptide is characterized by detecting the presence or absence of a genetic lesion characterized by at least one of these: (i) an alteration affecting the integrity of a gene encoding a marker polypeptides, or

(ii) the misexpression of the encoding polynucleotide.

To illustrate, such genetic lesions can be detected by ascertaining the existence of at least one of these:

1. a deletion of one or more nucleotides from the polynucleotide sequence

II. an addition of one or more nucleotides to the polynucleotide sequence

III. a substitution of one or more nucleotides of the polynucleotide sequence

IV. a gross chromosomal rearrangement of the polynucleotide sequence

V. a gross alteration in the level of a messenger RNA transcript of the polynucleotide sequence

VI. aberrant modification of the polynucleotide sequence, such as of the methylation pattern of the genomic DNA

VII. the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene

Viπ. a non- wild type level of the marker polypeptide

IX. allelic loss of the gene

X. inappropriate post-translational modification of the marker polypeptide

The present Invention provides assay techniques for detecting mutations in the encoding polynucleotide sequence. These methods include, but are not limited to, methods involving sequence analysis, Southern blot hybridization, restriction enzyme site mapping, and methods involving detection of absence of nucleotide pairing . between the polynucleotide to be analyzed and a probe.

Specific diseases or disorders, e.g., genetic diseases or disorders, are associated with specific allelic variants of polymoφhic regions of certain genes, which do not necessarily encode a mutated protein. Thus, the presence of a specific allelic variant of a polymorphic region of a gene in a subject can render the subject susceptible to developing a specific disease or disorder. Polymorphic regions in genes, can be identified, by determining the nucleotide sequence of genes in populations of individuals. If a polymorphic region is identified, then the link with a specific disease can be determined by studying specific populations of individuals, e.g. individuals which developed a specific disease, such as COPD. A polymorphic region can be located in any region of a gene, e.g., exons, in coding or non coding regions of exons, introns, and promoter region.

In an exemplary embodiment, there is provided a polynucleotide composition comprising a polynucleotide probe including a region of nucleotide sequence which is capable of hybridising to a sense or antisense sequence of a gene or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject genes or naturally occurring mutants thereof. The polynucleotide of a cell is rendered accessible for hybridization, the probe is contacted with the polynucleotide of the sample, and the hybridization of the probe to the sample polynucleotide is detected. Such techniques can be used to detect lesions or allelic variants at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.

A preferred detection method is allele specific hybridization using probes overlapping the mutation or polymorphic site and having about 5, 10, 20, 25, or 30 nucleotides around the mutation or polymorphic region. In a preferred embodiment of the invention, several probes capable of hybridising specifically to allelic variants are attached to a solid phase support, e.g., a "chip". Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA .probe arrays" is described e.g., in Cronin et al. (119). In one embodiment, a chip comprises all the allelic variants of at least one polymoφhic region of a gene. The solid phase support is the contacted with a test polynucleotide and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.

In certain embodiments, detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligase chain reaction (LCR) [Landegran et al., 1988, (120) and Nakazawa et al., 1994 (121)], the latter of which can be particularly useful for detecting point mutations in the gene; Abravaya et al., 1995 ,(122)]. In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating polynucleotide (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the polynucleotide sample with one or more primers which specifically hybridize to a polynucleotide sequence under conditions such that hybridization and amplification of the polynucleotide (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication [Guatelli, J.C. et al., 1990, (123)], transcriptional amplification system [Kwoh, D.Y. et al., 1989, (124)], Q-Beta replicase [Lizardi, P.M. et al., 1988 ,(125)], or any other polynucleotide amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of polynucleotide molecules if such molecules are present in very low numbers.

In a preferred embodiment of the subject assay, mutations in, or allelic variants, of a gene from a sample cell are identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis. Moreover; the use of sequence specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

4, In situ hybridization

In one aspect, the method comprises in situ hybridization with a probe derived from a given marker polynucleotide, which sequence is selected from any of the polynucleotide sequences of the SEQ ID NO: 1 or a sequence complementary thereto. The method comprises contacting the labeled hybridization probe with a sample of a given type of tissue from a patient potentially having COPD and COPD in particular as well as normal tissue from a person with no COPD, and determining whether the probe labels tissue of the patient to a degree significantly different (e.g., by at least a factor of two, or at least a factor of five, or at least a factor of twenty, or at least a factor of fifty) than the degree to which normal tissue is labeled.

Polypeptide detection

The subject invention further provides a method of determining whether a cell sample obtained from a subject possesses an abnormal amount of marker polypeptide which comprises (a) obtaining a cell sample from the subject, (b) quantitatively determining the amount of the marker polypeptide in the sample so obtained, and (c) comparing the amount of the marker polypeptide so determined with a known standard, so as to thereby determine whether the cell sample obtained from the subject possesses an abnormal amount of the marker polypeptide. Such marker polypeptide may be detected by immunohistochemical assays, dot-blot assays, ELISA and the like. Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of an MS4A8B polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of an MS4A8B polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form ah epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of an MS4A8B polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radio- immunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.

Typically, an antibody which specifically binds to an MS4A8B polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to MS4A8B polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate an MS4A8B polypeptide from solution.

• MS4A8B polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, an MS4A8B polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to an MS4A8B polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B- cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984).

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851- 6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such- antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to an MS4A8B polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to MS4A8B polypeptides. Antibodies with related specificity, but of distinct idiotypic composition. can be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton Proc. Nat Acad. Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11) Single-chain antibodies can be mono- or bispecific, and can be bivalent or tefravalent, Construction of tefravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced- directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J Immunol. Meth. 165, 81-91). A tibodies which specifically bind to MS4A8B polypeptides also can be produced by inducing in ^• vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can .be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which an MS4A8B polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Immunohistochemistry

Where tissue samples are employed, immunohistochemical staining may be used to determine the number of cells having the marker polypeptide phenotype. For such staining, a multiblock of tissue is taken from the biopsy or other tissue sample and subjected to proteolytic hydrolysis, employing such agents as protease K or pepsin. In certain embodiments, it may be desirable to isolate a nuclear fraction from the sample cells and detect the level of the marker polypeptide in the nuclear fraction.

^' The tissues samples are fixed by freatment with a reagent such as formalin, glutaraldehyde, methanol, or the like. The samples are then incubated with an antibody, preferably a monoclonal antibody, with binding specificity for the marker polypeptides. This antibody may be conjugated to a Label for subsequent detection of binding, samples are incubated for a time Sufficient for formation of the immunocomplexes. Binding of the antibody is then detected by virtue of a Label conjugated to this antibody. Where the antibody is unlabelled, a second labeled antibody may be employed, e.g., which is specific for the isotype of the anti-marker polypeptide antibody. Examples of labels which may be employed include radionuclides, fluorescence, chemilu- minescence, and enzymes.

Where enzymes are employed, the Substrate for the enzyme may be added to the samples to provide a colored or fluorescent product. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.

In one embodiment, the assay is performed as a dot blot assay. The dot blot assay finds particular application where tissue samples are employed as it allows determination of the average amount of the marker polypeptide associated with a Single cell by correlating the amount of marker ^' polypeptide in a cell-free extract produced from a predetermined number of cells.

In yet another embodiment, the invention contemplates using a panel of antibodies which are generated against the marker polypeptides of this invention, which polypeptides are encoded by the polynucleotide sequence of the SEQ ID NO: 1. Such a panel of antibodies may be used as a reliable diagnostic probe for COPD. The assay of the present invention comprises contacting a biopsy sample containing cells, e.g., macrophages, with a panel of antibodies to one or more of the encoded products to determine the presence or absence of the marker polypeptides.

The diagnostic methods of the subject invention may also be employed as follow-up to treatment, e.g., quantification of the level of marker polypeptides may be indicative of the effectiveness of current or previously employed therapies for COPD and COPD in particular as well as the effect of these therapies upon patient prognosis .

The diagnostic assays described above can be adapted to be used as prognostic assays, as well. Such an application takes advantage of the sensitivity of the assays of the Invention to events which take place at characteristic stages in the progression of plaque generation in case of COPD. For example, a given marker gene may be up- or down-regulated at a very early stage, perhaps before the cell is developing into a foam cell, while another marker gene may be characteristically up or down regulated only at a much later stage. Such a method could involve the steps of contacting the mRNA of a test cell with a polynucleotide probe derived from a given marker polynucleotide which is expressed at different characteristic levels in COPD tissue cells at different stages of COPD progression, and determining the approximate amount of hybridization of the probe to the mRNA of the cell, such amount being an indication of the level of expression of the gene in the cell, and thus an indication of the stage of disease progression of the cell; alternatively, the assay can be carried out with an antibody specific for the gene product of the given marker polynucleotide, contacted with the proteins of the test cell. A battery of such tests will disclose not only the existence of a certain arteriosclerotic plaque, but also will allow the clinician to select the mode of freatment most appropriate for the disease, and to predict the likelihood of success of that treatment. The methods of the invention can also be used to follow the clinical course of a given COPD predisposition. For example, the assay of the Invention can be applied to a blood sample from a patient; following treatment of the patient for COPD, another blood sample is taken and the test repeated. Successful treatment will result in removal of demonstrate differential expression, characteristic of the COPD tissue cells, perhaps approaching or even suφassing normal levels.

Polypeptide activity

In one embodiment the present invention provides a method for screening potentially therapeutic agents which modulate the activity of one or more "MS4A8B GENE" polypeptides, such that if the activity of the polypeptide is increased as a result of the upregulation of the "MS4A8B GENE" in a subject having or at risk for COPD and COPD in particular, the therapeutic substance will decrease the activity of the polypeptide relative to the activity of the some polypeptide in a subject not having or not at risk for COPD or COPD in particular but not treated with the therapeutic agent. Likewise, if the activity of the polypeptide as a result of the downregulation of the "MS4A8B GENE" is decreased in a subject having or at risk for COPD or COPD in particular, the therapeutic agent will increase the activity of the polypeptide relative to the activity of the same polypeptide in a subject not having or not at risk for COPD or COPD in particular, but not treated with the therapeutic agent.

The activity of the "MS4A8B GENE" polypeptides indicated in Table 2 or 3 may be measured by any means known to those of skill in the art, and which are particular for the type of activity performed by the particular polypeptide. Examples of specific assays which may be used to measure the activity of particular polynucleotides are shown below.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of MS4A8B gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non- phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphoro- dithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev. 90, 543-583, 1990.

Modifications of MS4A8B gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the MS4A8B. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base- pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of an MS4A8B polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an MS4A8B polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent MS4A8B nucleotides, can provide sufficient targeting specificity for MS4A8B mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular MS A8B polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to an MS4A8B polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozymes

^" Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 5.43-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of an MS4A8B polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the MS4A8B polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within an MS4A8B RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate MS4A8B RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated fransfection, elecfroporation, or calcium phosphate precipi- tation, can be used to introduce a ribozyrne-containing DNA construct into cells in which it is desired to decrease MS4A8B expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes ^" also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whose products interact with human MS4A8B. Such genes may represent genes which are differentially expressed in disorders including, but not limited to, diseases affecting the respiratory tissues such as chronic obstructive pulmonary disease.

Further, such genes may represent genes which are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes . may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human MS4A8B gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. . Any RNA isolation technique which does not select against the isolation of mRNA may be utilized for the purification of such RNA samples.. See, for example, Ausubel et al, ed.„ CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single- step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples which represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subfractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S.-. Patent 5,262,311), and microarrays.

• The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human MS4A8B. For example, freatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human MS4A8B. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human MS4A8B gene or gene product are up- regulated or down-regulated.

Promoter assays

A promoter assay was set up with a human hepatocellular carcinoma cell HepG2 that was stably transfected with a luciferase gene under the control of a gene of interest (e.g. thyroid hormone) regulated promoter. The vector 2xIR01uc, which was used for fransfection, carries a thyroid hormone responsive element (TRE) of two 12 bp inverted palindromes separated by an 8 bp spacer in front of a tk minimal promoter and the luciferase gene. Test cultures were seeded in 96 well plates in serum - free Eagle's Minimal Essential Medium supplemented with glutamine, tricine, sodium pyruvate, non - essential amino acids, insulin, selen, transferrin, and were cultivated in a humidified atmosphere at 10 % C0₂ at 37°C. After 48 hours of incubation serial dilutions of test compounds or reference compounds (L-T3, L-T4 e.g.) and co-stimulator if appropriate (final concentration 1 nM) were added to the cell cultures and incubation was continued for the optimal time (e.g. another 4-72 hours). The cells were then lysed by addition of buffer containing Triton X100 and luciferin and the luminescence of luciferase induced by T3 or other compounds was measured in a luminometer. For each concentration of a test compound replicates of 4 were tested. EC₅o - values for each test compound were calculated by use of the Graph Pad Prism Scientific software. Screening Methods

The invention provides assays for screening test compounds which bind to and/or modulate the activity of an MS4A8B polypeptide or an MS4A8B polynucleotide. A test compound preferably binds to an MS4A8B polypeptide or polynucleotide. More preferably, a test compound decreases or increases a biological effect mediated via human MS4A8B by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc, Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to MS4A8B polypeptides or polynucleotides or to affect MS4A8B activity or MS4A8B gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jay^~awickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in pefri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is^' described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together. Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site of the MS4A8B polypeptide, thereby making the ligand binding site inaccessible to substance such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the MS4A8B polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the MS4A8B polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate subsfrate to a detectable product.

Alternatively, binding of a test compound to an MS4A8B polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with an MS4A8B polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument. that measures the rate- at which a cell acidifies its environment using a light- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and an MS4A8B polypeptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to an MS4A8B polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, an MS4A8B polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Barrel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the MS4A8B polypeptide and modulate its activity. The two-hybrid system is based on the modular nature of most franscription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding an MS4A8B polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the franscription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional franscription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the MS4A8B polypeptide.

It may be desirable to immobilize either the MS4A8B polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the MS4A8B polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the MS4A8B polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absoφtion, or pairs of binding moieties attached respectively to the polypeptide

(or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to an MS4A8B polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the. reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the MS4A8B polypeptide is a fusion protein comprising a domain that allows the MS4A8B polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto> glutathione sepharose beads (Sigma Chemical) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed MS4A8B polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before ^binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either an MS4A8B polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated MS4A8B polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of sfreptavidin- coated 96 well plates (Pierce 'Chemical). Alternatively, antibodies which specifically bind to an MS4A8B polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the MS4A8B polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST- immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the MS4A8B polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the MS4A8B polypeptide, and SDS gel electrophoresis under non- reducing conditions.

Screening for test compounds which bind to an MS4A8B polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises an MS4A8B polypeptide or polynucleotide can be used in a cell-based assay system. Art MS4A8B polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to an MS4A8B polypeptide or polynucleotide is determined as described above.

Functional Assays

Test compounds can be tested for the ability to increase or decrease a biological effect of an MS4A8B polypeptide. Such biological effects can be determined using functional assays such as those described in the specific examples, below. Functional assays can be carried out after contacting either a purified MS4A8B polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which decreases an activity of an MS4A8B by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) is identified as a potential therapeutic agent for decreasing MS4A8B activity. A test compound which increases an MS4A8B activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing MS4A8B activity.

One such screening procedure involves the use of melanophores which are transfected to express an MS4A8B polypeptide. Such a screening technique is described in WO 92/01810 published Feb. 6, 1992. Thus, for example, such an assay may be employed for screening for a compound which inhibits activation of the receptor polypeptide by contacting the melanophore cells which comprise the receptor with both a receptor ligand and a test compound to be screened. Inhibition of the signal generated by the ligand indicates that a test compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor. The screen may be employed for identifying a test compound which activates the receptor by contacting such cells with compounds to be screened and determining whether each test compound generates a signal, i.e., activates the receptor.

Other screening techniques include the use of cells which express a human MS4A8B polypeptide (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation (see, e.g., Science 246, 181-296, 1989). For example, test compounds may be contacted with a cell which expresses a human MS4A8B polypeptide and a second messenger response, e.g., signal fransduction or pH changes, can be measured to determine whether the test compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding a human MS4A8B polypeptide into Xenopus oocytes to transiently express the receptor. The transfected oocytes can then be contacted with the receptor ligand and a test compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for test compounds which are thought to inhibit activation of the receptor.

Another screening technique involves expressing a human MS4A8B polypeptide in cells in which the receptor is linked to a phospholipase C or D. Such cells include endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening may be accomplished as described above by quantifying the degree of activation of the receptor from changes in the phospholipase activity.

Details of functional assays such as those described above are provided in the specific examples, below.

Gene Expression

In another embodiment, test compounds which increase or decrease MS4A8B gene expression are identified. An MS4A8B polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the MS4A8B polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of MS4A8B mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of an MS4A8B polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incoφoration of labeled amino acids into an MS4A8B polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses an MS4A8B polynucleotide can be used in a cell-based assay system. The MS4A8B polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or hunian embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions which can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, an MS4A8B polypeptide, MS4A8B polynucleotide, antibodies which specifically bind to an MS4A8B polypeptide, or mimetics, agonists, antagonists, or inhibitors of an MS4A8B polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dexfrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, infrathecal, infraventricular, transdermal, subcutaneous, infraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets; pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active com-_, pounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings,, such as concenfrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or friglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penefrants appropriate to the particular barrier to be permeated are used in the formulation. Such penefrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for freatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Ther'apeutic Indications and Methods

The human MS4A8B of the invention can be regulated to treat diseases affecting the respiratory tissues, such as chronic obstructive pulmonary disease. This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or an MS4A8B polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. ■

COPD. Chronic obstructive pulmonary (or airways) disease (COPD) is a condition defined physiologically as airflow obstruction that generally results from a mixture of emphysema and peripheral airway obstruction due to chronic bronchitis (Senior & Shapiro, Pulmonary Diseases

^■ and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp. 659-681, 1998; Barnes, Chest 117,

10S-14S, 2000). Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung. Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years. In COPD, airflow obstruction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does occur in non-smokers.

Chronic inflammation of the airways is a key pathological feature of COPD (Senior & Shapiro, 1998). The inflammatory cell population comprises increased numbers of macrophages, neutrophils, and CD8⁺ lymphocytes. Inhaled irritants, such as cigarette- smoke, activate macrophages which are resident in the respiratory tract, as well as epithelial cells leading to release of chemokines (e.g., interleukin- 8) and other chemotactic factors. These chemotactic factors act to increase the neutrophil/monocyte trafficking from the blood into the lung tissue and airways. Neufrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species. Matrix degradation and emphysema, along with airway wall thickening, surfactant dysfunction, and mucus hypersecretion, all are potential sequelae of this inflammatory response that lead to impaired airflow and gas exchange.

A reagent which affects MS4A8B activity can be administered to a human cell, either in vitro or in vivo, to reduce MS4A8B activity. The reagent preferably binds to an expression product of a human MS4A8B gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the fransfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell types, such as a cell-specific ligand exposed on the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor- mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991). Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases MS4A8B activity relative to the MS4A8B activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and^' routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀ ED₅₀.

Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires freatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation. Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.- Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA fransfer, fransfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, elecfroporation, "gene gun," and DEAE- or calcium phosphate-mediated fransfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single- chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of an MS4A8B gene¹ or the activity of an MS4A8B polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of an MS4A8B gene or the activity of an MS4A8B polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to MS4A8B- specific mRNA, quantitative RT-PCR, immunologic detection of an MS4A8B polypeptide, or measurement of MS4A8B activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Diagnostic Methods

Human MS4A8B also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode a MS4A8B. Such diseases, by way of example, include COPD.

According to the present invention, differences can be determined between the cDNA or genomic sequence encoding MS4A8B in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85, 4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-elecfrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of an MS4A8B also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays. All patents and patent applications cited in this disclosure are expressly incoφorated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided for puφoses of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1

Effect of a test compound on the mobilization of intracellular calcium

Intracellular free calcium concentration can be measured by microspectrofluorometry using the fluorescent indicator dye Fura-2/AM (Bush et al, J. Neurochem. 57, 562-74, 1991). Stably transfected cells are seeded onto a 35 mm culture dish containing a glass coverslip insert. Cells are washed with HBS , incubated with a test compound, and loaded with 100 μl of Fura-2/AM (10 μM) for 20-40 minutes. After washing with HBS to remove the Fura-2/AM^' solution, cells are equilibrated in HBS for 10-20 minutes. Cells are then visualized under the 40X objective of a Leitz Fluovert FS microscope.

Fluorescence emission is determined at 510 nM, with excitation wavelengths alternating between 340 nM and 380 nM. Raw fluorescence data are converted to calcium concentrations using standard calcium concenfration curves and software analysis techniques. A test compound which increases the fluorescence by at least 15% relative to fluorescence in the absence of a test compound is identified as a compound which mobilizes intracellular calcium.

EXAMPLE 2

Expression of recombinant human MS4A8B

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human GPCR polypeptides in yeast. The GPCR-encoding DNA sequence is derived from SEQ ID NO: 1. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invifrogen, San Diego, CA) according to manufacturer's instructions. Purified human GPCR polypeptide is obtained.

EXAMPLE 3

Identification of test compounds that bind to MS4A8B polypeptides

Purified MS4A8B polypeptides comprising a glutathione-S-fransferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. MS4A8B polypeptides comprise an amino acid sequence shown in SEQ ID NO: 2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a MS4A8B polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a MS4A8B polypeptide.

EXAMPLE 4

Identification of a test compound which increases MS4A8B gene expression

A test compound is administered to a culture of human gastric cells and incubated at 37°C for 10 . to 45 minutes. A culture of the same type of cells incubated for the same time without the test compound provides a negative confrol.

RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled MS4A8B-specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1. A test compound which increases the MS4A8B-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of MS4A8B gene expression. EXAMPLE 5

Identification of a test compound which decreases MS4A8B gene expression

A test compound is administered to a culture of human gastric cells and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells incubated for the same time without the test compound provides a negative confrol.

RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled MS4A8B-specific probe at 65°^°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1. A test compound which decreases the MS4A8B-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of _.MS4A8B gene expression.

EXAMPLE 6

Treatment of COPD -with a reagent which specifically binds to an MS4A8B gene product

Synthesis of antisense MS4A8B oligonucleotides comprising at least 11 contiguous nucleotides selected from the complement of SEQ ID NO. 1 is performed on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure (Uhlmann et al, Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concenfration. Purity of these oligonucleotides is tested by capillary gel elecfrophoreses and ion exchange HPLC. Endotoxin levels in the oligonucleotide preparation are determined using the Luminous Amebocyte Assay (Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953). The antisense oligonucleotides are administered intrabronchially to a patient with COPD. The severity of the patient's COPD is decreased.

EXAMPLE 7

Disease-specific expression ofMS4A8B detected by microarray analysis

Target preparation

Human total RNA was prepared from frozen lung tissue obtained from two normal individuals and two individuals diagnosed with chronic obstructive pulmonary disease (Analytical Biological Services Inc. Wilmington, DE, USA) using Trizol™ (Invifrogen Coφ., Carlsbad, CA, USA). Five micrograms of each of the total RNAs was. added to a reaction mix in a final volume of 12 μl, containing bacterial confrol mRNAs (2.5 pg/μl araB/entF, 8.33 pg/μl fϊxB/gnd and 25 pg/μl hisB/leuB) and 1.0 μl of 0.5 pmol/μl T7-(dT)₂₄ oligonucleotide primer. The mixture was incubated for 10 min at 70°C and chilled on ice. With the mixture remaining on ice, 4 μl of 5x first-strand buffer, 2 μl 0.1 M DTT, 1 μl of 10 mM dNTP mix and 1 μl Superscript™ II RNase H- reverse transcriptase (200 U/μl) was added to make a final volume of 20 μl, and the mixture incubated for 1 h in a 42°C water bath. Second-strand cDNA was synthesized in a final volume of 150 μl, in a mixture containing 30 μl of 5x second-strand buffer, 3 μl of 10 mM dNTP mix, 4 μl of Escherichia coli DNA polymerase I (10 U/μl) and 1 μl of RNase H (2 U/μl) for 2 h at 16°C. The cDNA was purified using a Qiagen QIAquick purification kit, dried down, and resuspended in IVT reaction mix, containing 3.0 μl nuclease-free water, 4.0 μl lOx reaction buffer, 4.0 μl 75 mM ATP, 4.0 μl 75 mM GTP, 3.0 μl 75 mM CTP, 3.0 μl 75 mM UTP, 7.5 μl 10 mM- Biotin 11-CTP, 7.5 μl 10 mM Biotin 11-UTP (PerkinElmer Life Sciences Inc. Boston, MA, USA) and 4.0 μl enzyme mix. The reaction mix was incubated for 14 h at 37 °C and cRNA target purified using an RNeasy^® kit (Qiagen). cRNA yield was quantified by measuring the UV absorbance at 260 nm, and fragmented in 40 mM Tris-acetate (TrisOAc) pH 7.9, 100 mM KOAc and 31.5 mM MgOAc, at 94°C for 20 min. This results typically in a fragmented target with a size range between 100 and 200 bases.

Array hybridization

Ten micrograms of fragmented target cRNA was used for hybridization of each UniSet Human I Expression Bioarray chip(AmershamBiosciences), in 260 μl of hybridization solution containing 78 μl Amersham Hyb buffer component A and 130 μl Amersham Hyb buffer component B. The hybridization solution was heated at 90°C for 5 min to denature the cRNA and chilled on ice. The sample was vortexed for 5 s at maximum speed, and 250 μl injected into the inlet port of the hybridization chamber. The slides were loaded into a ISF-4-W shaking incubator(Kuhner, Birsfelden, Switzerland), with the hybridization chambers facing up. Slides were incubated for 24 h at 37°C, while shaking at 300 r.p.m.

Post-hybridization processing using streptavidin-Cy5

The slides were removed from the ISF-4-W shaker, and the hybridization chamber removed from each slide. Each slide was briefly rinsed in TNT buffer (0.1 M Tris-HCl pH 7.6, 0.15 M NaCl, 0.05% Tween-20) at room temperature, and then washed in TNT buffer at 42 °C for 60 min. The signal was developed using a 1:500 dilution of streptavidin-Cy5 (AmershamBiosciences), for 30 min at room temperature. Excess dye was removed by washing four times with TNT buffer, for 5 min each, at room temperature. Slides were rinsed in 0.05% Tween-20 and dried under nitrogen gas. Processed slides were scanned using an Axon GenePix 4000B Scanner with the laser set to 635 nm, the photomultiplier tube (PMT) voltage to 600 and the scan resolution to lOμm. Images were acquired with the Axon GenePixPro v4.0 Scanning Software (AmershamBiosciences), and analyzed using the CodeLink™ Expression Analysis Software (AmershamBiosciences).

Data analysis

CodeLink™ Expression Analysis Software(AmershamBiosciences) automatically creates signal data for each spotted dot as a Microsoft Excel formatted spreadsheet. The data was then compared using the computer program Spotfire Decision Site 7.0 (Spotfire Japan K.K., Tokyo, Japan) to determine the fold difference between each gene in normal and COPD lung. As a result of this analysis, the MS4A8B gene transcript was found to be expressed higher in COPD than in normal lung.

EXAMPLE 8

Tissue-specific expression ofMS4A8B

The qualitative expression pattern of MS4A8B in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

To demonstrate that MS4A8B is involved in the disease process of COPD, 25 μg of total RNA from the following sources were used as template in reactions to synthesize first-strand cDNA for expression profiling : Human Total RNA Panel I-V (Clontech Laboratories, Palo Alto, CA, USA), normal human lung primary cell lines (BioWhittaker Clonetics, Walkersville, MD, USA), human umbilical vein endothelial cells (HUVECs) (Kurabo, Osaka, Japan), several common cell lines (ATCC, Washington, DC), and various cells purified from peripheral blood. First-strand cDNA was synthesized using oligo (dT) (Nippon Gene Research Laboratories, Sendai, Japan) and the SUPERSCRIPT™ First-Strand Synthesis System for RT-PCR (Life Technologies, Rockville, MD) according to the manufacturer's protocol. For these samples, 1/1250* of the synthesized first- strand cDNA was subsequently used as template for quantitative PCR. Additional samples were purchased as presynthesized cDNAs (Human Immune System MTC Panel and Human Blood Fractions MTC Panel, Clontech Laboratories), and for these, 10 ng of cDNA was used as template for quantitative PCR.

Quantitative PCR was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN) with oligonucleotide primers 5'-TTCGGATCGAATCTCGCCTGCT-3' and 5'- TGCTTGCTCAAGGTTCCCGCTTA-3' in the presence of the DNA-binding fluorescent dye SYBR Green I. Results were then converted into copy numbers of the gene transcript per ng of template cDNA by fitting to a standard curve. The standard curve was derived by simultaneously performing the quantitative PCR reaction on PCR products of known concentrations amplified beforehand from the target gene.

To correct for differences in mRNA franscription levels per cell in the various tissue types, a normalization procedure was performed using similarly calculated expression levels of five different housekeeping genes: glyceraldehyde-3 -phosphatase (GAPDH), hypoxanthine guanine phophoribosyl fransferase (HPRT), beta-actin, poφhobilinogen deaminase (PBGD), and beta-2- microglobulin. The level of housekeeping gene expression is considered to be relatively constant for all tissues (Adams et al., 1993, Adams et al., 1995, Liew et al., 1994) and therefore can be used as a gauge to approximate relative numbers of cells per ng of cDNA template. Expression levels of the five housekeeping genes in all tissue samples were measured in three independent reactions per gene using the LightCycler and a constant amount (25 μg) of starting RNA. The calculated copy numbers for each gene, derived from comparison with simultaneously reacted standards of known concentrations, were recorded and converted into a percentage of the , sum of the copy numbers of the gene in all tissue samples. For each tissue sample, the sum of the percentage values for each gene was calculated, and a normalization factor was calculated by dividing the sum percentage value for each tissue by the sum percentage value of one of the tissues arbitrarily selected as a standard. To normalize an experimentally obtained value for the expression of a particular gene in a tissue sample, the obtained value was multiplied by the normalization factor for the tissue tested. This normalization method was used for all tissues except those derived from the Human Blood Fractions MTC Panel, which were normalized against the single housekeeping gene, beta-2-microglobulin, due to wide variation in other housekeeping gene expression in these tissues depending on activation status. The results of this expression profiling are given in Figure 9 and Figure 10, showing the normalized values for the copy numbers of mRNA per 10 ng of first- strand cDNA in each sample tested.

To measure the relative copy numbers of the genes in patient samples and healthy lung samples, total RNA was prepared from frozen lung tissue obtained from two normal individuals and two individuals diagnosed with chronic obstructive pulmonary disease (Analytical Biological Services Inc. Wilmington, DE, USA) using Trizol™ (Invifrogen Corp., Carlsbad, CA, USA). cDNA was then synthesized as above and used as template for quantitative PCR as described above. Normalization was performed using the single housekeeping gene GAPDH. The different levels of expression of the MS4A8B gene transcript between the normal and COPD lung tissue samples are shown in Figure 8, which displays the ratio of MS4A8B transcript to GAPDH transcript measured in each sample tested.

Claims

1. A method for the prediction, diagnosis or prognosis of respiratory diseases by the detection of expression level of the MS4A8B gene or genomic nucleic acid sequences.

2.- The method of claim 1 wherein the respiratory disease is COPD.

3. The method of claim 1 or 2 wherein the detection method comprises the use of PCR, arrays or beads.

4. A method for the prediction, diagnosis or prognosis of COPD by the detection of at least one marker characterized in that at least 1 marker is selected from:

a) a polynucleotide or polynucleotide analog comprising the sequences of SEQ ID NO: 1;

b) a polynucleotide or polynucleotide analog which hybridizes under stringent conditions to a polynucleotide specified in (a) and encodes a polypeptide exhibiting the same biological function as MS4A8B;

c) a polynucleotide or polynucleotide analog, the sequence of which deviates from the polynucleotide specified in (a) and (b) due to. the degeneracy of the genetic code, encoding a polypeptide exhibiting the same biological function as MS4A8B;

d) a polynucleotide or polynucleotide analog which represents a specific fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (c) encoding a polypeptide exhibiting the same biological function as MS4A8B;

e) a purified polypeptide encoded by a polynucleotide or polynucleotide analog sequence specified in (a) to (d);

f) a purified polypeptide comprising at least one of the sequences of SEQ ID NO: 2;

are detected.

5. A method for the prediction, diagnosis or prognosis of COPD by the detection of at least 2 markers characterized in that at least 2 markers are selected from:

a) a polynucleotide or polynucleotide analog comprising the sequence of SEQ ID

NO: 1; b) a polynucleotide or polynucleotide analog which hybridizes under stringent conditions to a polynucleotide specified in (a) and encodes a polypeptide exhibiting the same biological function as MS4A8B;

c) a polynucleotide or polynucleotide analog the sequence of which deviates from the polynucleotide specified in (a) and (b) due to the generation of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B;

e) a purified polypeptide encoded by a polynucleotide sequence or polynucleotide analog specified in (a) to (d);

f) a purified polypeptide comprising the sequence of SEQ ID NO: 2;

are detected.

6. A diagnostic kit for conducting the method of anyone of claims 1 to 5.

7. A composition for the prediction, diagnosis or prognosis of COPD comprising:

a) a detection agent for:

i. a polynucleotide or polynucleotide analog comprising at least one of the sequence of SEQ ID NO: 1 ;

ii. any polynucleotide or polynucleotide analog which hybridizes under stringent conditions to a polynucleotide specified in (i) encoding a polypeptide exhibiting the same biological function as MS4A8B;

iii. a polynucleotide or polynucleotide analog the sequence of which deviates from the polynucleotide specified in (i) and (ii) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological < function as MS4A8B;

iv. a polynucleotide or polynucleotide analog which represents a specific fragment, derivative or allelic variation of a polynucleotide sequence specified in (i) to (iii) encoding a polypeptide exhibiting the same biological function as MS4A8B;

v. a polypeptide encoded by a polynucleotide or polynucleotide analog sequence specified in (i) to (iv);

^' vi. a polypeptide comprising at least one of the sequences of SEQ ID NO: 2.

or -

b) at least 2 detection agents for at least 2 markers selected from:

i. any polynucleotide comprising at least one of the sequences of SEQ ID

NO: 1;

ii. any polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (i) encoding a polypeptide exhibiting the same biological function as MS4A8B;

iii. a polynucleotide the sequence of which deviates from the polynucleotide specified in (i) and (ii) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B;

iv. a polynucleotide which represents a specific fragment, derivative or allelic variation of a polynucleotide sequence specified in (i) to (iii) encoding a polypeptide exhibiting the same biological function as MS4A8B;

v. a polypeptide encoded by a polynucleotide sequence specified in (i) to (iv);

vi. a polypeptide comprising at least one of the sequences of SEQ ID NO: 2.

8. An array comprising a plurality of polynucleotides or polynucleotide analogs wherein each of the polynucleotides is selected from:

a) a polynucleotide or polynucleotide analog comprising at least one of the sequences of SEQ ID NO: 1;

b) a polynucleotide or polynucleotide analog which hybridizes under sfringent conditions to a polynucleotide specified in (a) encoding a polypeptide exhibiting

' the same biological function as MS4A8B; c) a polynucleotide or polynucleotide analog the sequence of which deviates from the polynucleotide specified in (a) and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B;

attached to a solid support.

9. A method of screening for agents which regulate the activity of a polypeptide encoded by a polynucleotide or polynucleotide analog selected from the group consisting of:

a) a polynucleotide or polynucleotide analog comprising the sequence of SEQ ID

NO: 1;

b) a polynucleotide or polynucleotide analog which hybridizes under sfringent conditions to a polynucleotide specified in (a) encoding a polypeptide exhibiting the same biological function as MS4A8B;

c) a polynucleotide or polynucleotide analog the sequence of which deviates from the polynucleotide specified in (a) and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B;

comprising the steps of:

i. contacting a test compound with at least one polypeptide encoded by a polynucleotide specified in (a) to (d); and

ii. detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for modulating the activity of the polypeptide in order to prevent of treat COPD.

10. A method of screening for agents which regulate the activity of a polypeptide encoded by a polynucleotide or polynucleotide analog selected from the group consisting of: a) a polynucleotide or polynucleotide analog comprising the sequences of SEQ ID NO: 1;

comprising the steps of:

ii. detecting the activity of the polypeptide as MS4A8B, wherein a test compound which increases the activity is identified as a potential preventive or therapeutic agent for increasing the polypeptide activity in COPD, and wherein a test coin- pound which decreases the activity of the polypeptide is identified as a potential therapeutic agent for decreasing the polypeptide activity in COPD.

11. ^' A method of screening for agents which regulate the activity of a polynucleotide or polynucleotide analog selected from group consisting of;

b) a polynucleotide or polynucleotide analog which hybridizes under sfringent condi- tions to a polynucleotide specified in (a) encoding a polypeptide exhibiting the same biological function as MS4A8B;

c) a polynucleotide or polynucleotide analog the sequence of which deviates from the polynucleotide specified in (a) and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B; d) a polynucleotide or polynucleotide analog which represents a specific fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (c) encoding a polypeptide exhibiting the same biological function as MS4A8B;

comprising the steps of:

i. contacting a test compound with at least one polynucleotide or polynucleotide analog specified in (a) to (d), and

ii. detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential preventive or therapeutic agent for regulating the activity of the polynucleotide in COPD.

12. Use of

b) a polynucleotide which hybridizes under stringent conditions to a polynucleotide or polynucleotide analog specified in (a) encoding a polypeptide exhibiting the same biological function as MS4A8B;

e) an antisense molecule targeting specifically one of the polynucleotide sequences specified in (a) to (d);

f) a purified polypeptide encoded by a polynucleotide or polynucleotide analog sequence specified in (a) to (d)

g) a purified polypeptide comprising at least one of the sequences of SEQ ID NO: 2;

h) an antibody capable of binding to one of the polynucleotide specified in (a) to (d) or a polypeptide specified in (f) and (g); i) a reagent identified by any of the methods of claim 14 to 16 that modulates the amount or activity of a polynucleotide sequence specified in (a) to (d) or a polypeptide specified in (f) and (g);

in the preparation of a composition for the prevention, prediction, diagnosis, prognosis or a medicament for the freatment of COPD.

13. Use of claim 12 wherein the disease is COPD.

14. A reagent that regulates the activity of a polypeptide selected from the group consisting of:

a) a polypeptide encoded by any polynucleotide or polynucleotide analog comprising at least one of the sequences of SEQ ID NO: 1;

b) a polypeptide encoded by any polynucleotide or polynucleotide analog which hybridizes under stringent onditions to any polynucleotide comprising the sequence of SEQ ID NO: 1 encoding a polypeptide exhibiting the same biological function as MS4A8B;

c) a polypeptide encoded by any polynucleotide or polynucleotide analog the sequence of which deviates from the polynucleotide specified in (a) and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting the same biological function as MS4A8B;

d) a polypeptide encoded by any . polynucleotide or polynucleotide analog which represents a specific fragment, derivative or εtllelic variation of a polynucleotide sequence specified in (a) to (c) encoding a polypeptide exhibiting the same biological function as MS4A8B;

e) or a polypeptide comprising at least one of the sequence of SEQ ID NO: 2

wherein said reagent is identified by the method of any of the claims 9 to 11.

15. A reagent that regulates the activity of a polynucleotide or polynucleotide analog selected from the group consisting of:

a) a polynucleotide or polynucleotide analog comprising the sequence SEQ ID

NO: 1; b) a polynucleotide or polynucleotide analog which hybridizes under stringent conditions to a polynucleotide specified in (a) encoding a polypeptide exhibiting the same biological function as MS4A8B;

d) a polynucleotide or polynucleotide analog which represents a specific fragment; derivative or allelic variation of a polynucleotide sequence specified in (a) to (c) encoding a polypeptide exhibiting the same biological function as MS4A8B;

wherein said reagent is identified by the method of any of the claims 9 to 11.

16. A pharmaceutical composition, comprising:

a) an expression vector containing at least one polynucleotide or polynucleotide analog selected from the group consisting of:

i. . a polynucleotide or polynucleotide analog comprising the sequence of SEQ ID NO: 1;

ii. a polynucleotide or polynucleotide analog which hybridizes under

• sfringent conditions to a polynucleotide specified in (a) encoding a polypeptide exhibiting the same biological function as MS4A8B;

iii. a polynucleotide or polynucleotide analog the sequence of which deviates from the polynucleotide specified in (a) and (b) due to the degeneracy of the genetic code_encoding a polypeptide exhibiting the same biological function as MS4A8B;

iv. a polynucleotide or polynucleotide analog which represents a specific fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (c)_encoding a polypeptide exhibiting the same biological function as MS4A8B;

or the reagent of claim 14 or 15 and a pharmaceutically acceptable carrier.

17. A computer-readable medium comprising: a) at least one digitally encoded value representing a level of expression of polynucleotide sequence of SEQ ED NO: 1;

b) in a cell from the a subject at risk for or having COPD.