Examples for chronic inflammatory airway diseases, in which macrophages play an important role is chronic bronchitis (CB). CB may occur with or without airflow limitation and includes chronic obstructive pulmonary disease (COPD). CB is a complex disease encompassing symptoms of several disorders: chronic bronchitis which is characterized by cough and mucus hypersecretion, small airway disease, including inflammation and peribronchial fibrosis, emphysema, and airflow limitation.

CB is characterized by an accelerated and irreversible decline of lung function. The major risk factor for developing CB is continuous cigarette smoking. Since only about 20% of all smokers are inflicted with CB, a genetic predisposition is also likely to contribute to the disease.

The initial events in the early onset of CB are inflammatory, affecting small and large airways. An irritation caused by cigarette smoking attracts macrophages and neutrophils the number of which is increased in the sputum of smokers. Perpetual smoking leads to an ongoing inflammatory response in the lung by releasing mediators from macrophages, neutrophils and epithelial cells that recruit inflammatory cells to sites of the injury. So far there is no therapy available to reverse the course of CB. Smoking cessation may reduce the decline of lung function.

Only a few drugs are known to date to provide some relief for patients. Long-lasting p2-agonists and anticholinergics are applied to achieve a transient bronchodilation. A

variety of antagonists for inflammatory events are under investigation like, LTB4- inhibitors.

There is a continuous need to provide drugs for treating chronic inflammatory airway diseases. Chronic inflammatory airway diseases can be attributed to activated inflammatory immune cells, e. g. macrophages. There is therefore a need for drugs modulating the function of macrophages in order to eliminate a source of inflammatory processes.

Description Of The Invention In the present invention it was found that macrophages involved in an inflammatory process, particularly in a chronic inflammatory airway disease, more particularly in chronic bronchitis or COPD, show a pattern of differentially expressed nucleic acid sequence and protein expression which differs from the pattern of gene expression of macrophages from healthy donors or donors in an irritated state, which latter do contain macrophages in an activated state. Therefore, macrophages show different activation levels under different inflammatory conditions. For example, it is shown in the present invention that macrophages involved in an inflammatory process in COPD smokers show different gene expression pattern than macrophages from healthy smokers, indicating that in COPD smokers macrophages are in a different, hereinafter named"hyperactivated"state. The present invention provides for the possibility to inhibit the hyperactivation or to reduce the hyperactive state of a macrophage by allowing the identification of substances which modulate a protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase, all depicted in the Sequence Listing hereinafter, involved in the hyperactivation or maintaining the hyperactive state of a macrophage.

The term"chronic inflammatory airway disease"as used hereinafter includes, for example, Chronic Bronchitis (CB) and Chronic Obstructive Pulmonary Disease (COPD). The preferred meaning of the term"chronic inflammatory airway disease"is CB and COPD, the more preferred meaning is CB or COPD.

The invention is based on the identification of a nucleic acid sequence differentially expressed in a hyperactivated macrophage compared to a macrophage which is not hyperactivated. Such a nucleic acid sequence encodes a protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA- Desaturase and UDP-Glucose Ceramide Glycosyltransferase, which protein is involved in the hyperactivation or maintaining the hyperactive state of a macrophage involved in an inflammatory process, preferably in a chronic inflammatory airway disease. Such differentially expressed nucleic acid sequence or protein encoded by such nucleic acid sequence is in the following also named differentially expressed nucleic acid sequence or protein of the invention, respectively. In particular, the present invention teaches a link between phenotypic changes in macrophages due to differentially expressed nucleic acid sequence and protein expression pattern and involvement of macrophages in inflammatory processes and, thus, provides a basis for a variety of applications. For example, the present invention provides a method and a test system for determining the expression level of a macrophage protein of the invention or differentially expressed nucleic acid sequence of the invention and thereby provides e. g. for methods for diagnosis or monitoring of inflammatory processes with involvement of hyperactivated macrophages in mammalian, preferably human beings, especially such beings suffering from an inflammatory process, preferably in a chronic inflammatory airway disease. The invention also relates to a method for identifying a substance by means of a differentially expressed nucleic acid sequence or protein of the invention, which substance modulates, i. e. acts as an inhibitor or activator on the said differentially expressed nucleic acid sequence or protein of the invention and thereby positively influences chronic inflammatory processes by inhibition of the hyperactivation or reduction of the hyperactive state of macrophages, and thereby allows treatment of mammals, preferably human beings, suffering from a said disease. The invention also relates to a method for selectively modulating such a differentially expressed nucleic acid sequence or protein of the invention in a macrophage comprising administering a substance determined to be a modulator of said protein or differentially expressed nucleic acid sequence. The present invention includes the use of said substances for

treating beings in need of a treatment for an inflammatory process, preferably a chronic inflammatory airway disease.

In the present invention in a first step a differentially expressed nucleic acid sequence of the invention is identified which has a different expression pattern in a hyperactivated macrophage compared to a macrophage which is not hyperactivated.

For the sake of conciseness this description deals particularly with investigation of macrophages involved in COPD, however, equivalent results may be obtained with samples from subjects suffering from other chronic inflammatory airway diseases, e. g. other chronic bronchitis symptoms. The investigation of the different expression pattern leads to the identification of a series of differentially expressed nucleic acid sequences expressed in dependency on the activation state of a macrophage involved in an inflammatory process, as exemplified in the Examples hereinbelow.

Briefly, such a differentially expressed nucleic acid sequence of the invention is identified by comparative expression profiling experiments using a cell or cellular extract from a hyperactivated macrophage, i. e. for example from the site of inflammation in COPD and from the corresponding site of control being not suffering from said disease, however, suffering under the same irritating condition like cigarette smoke exposure.

In a second step the proteins are identified which are encoded by the differentially expressed nucleic acid sequence, i. e. proteins playing a role in mediating the hyperactivation or in maintaining the hyperactivated state. A group of differentially expressed nucleic acid sequences of the invention can be identified to encode a protein which is selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase. A said protein is involved in the hyperactivation or maintaining the hyperactive state which is characterized in that it is expressed in a macrophage that is hyperactivated according the invention at a lower or higher level than the control level in a macrophage which is not hyperactivated.

Accordingly, the invention concerns a protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP- Glucose Ceramide Glycosyltransferase. A protein selected from the said group is hereinafter also named protein of the invention. The said proteins of the invention are depicted hereinafter in the Sequence Listing.

The biological activity of MIF (SEQ ID NO. 1,2) according to the present invention, i. e. mediating the involvement of a macrophage in an inflammatory process according to the invention, e. g. by inhibition of macrophage migration, is dependent, for example, on counteracting suppressive effects of glucocorticoids and/or on another MIF function like inducing inflammatory response to invasion of bacteria or any other function of MIF relevant for its biological activity according to the invention.

The invention also concerns a functional equivalent, derivative, variant, mutant or fragment of MIF. Functional in this context means having a function of the MIF that is involved in its biological activity according to the invention.

The biological activity of DAD1 (SEQ ID NO. 3,4) according to the present invention, i. e. mediating the involvement of a macrophage in an inflammatory process according to the invention, is dependent, for example, on binding to an oligosaccaryltransferase complex and/or on any other DAD1 function relevant for its biological activity according to the invention.

The invention also concerns a functional equivalent, derivative, variant, mutant or fragment of DAD1. Functional in this context means having a function of DAD1 that is involved in its biological activity according to the invention.

The biological activity of ARL4 (SEQ ID NO. 5,6) according to the present invention, i. e. mediating the involvement of a macrophage in an inflammatory process according to the invention, is dependent, for example, on interaction with proteins involved in vesicular and membrane trafficking and/or on any other ARL4 function relevant for its biological activity according to the invention.

The invention also concerns a functional equivalent, derivative, variant, mutant or fragment of ARL4. Functional in this context means having a function of ARL4 that is involved in its biological activity according to the invention.

The biological activity of GNS (SEQ ID NO. 7,8) according to the present invention, i. e. mediating the involvement of a macrophage in an inflammatory process according to the invention, is dependent, for example, on binding and/or recognizing a substrate, e. g. heparan and/or on its hydrolytic activity and/or on any other GNS function relevant for its biological activity according to the invention.

The invention also concerns a functional equivalent, derivative, variant, mutant or fragment of GNS. Functional in this context means having a function of GNS that is involved in its biological activity according to the invention.

The biological activity of Transglutaminase 2 (SEQ ID NO. 9,10) according to the present invention, i. e. mediating the involvement of a macrophage in an inflammatory process according to the invention, is dependent, for example, on formation of (y- glutamyl) lysine isopeptide bonds and/or on any other Transglutaminase 2 function, e. g. substrate recognition, relevant for its biological activity according to the invention.

The invention also concerns a functional equivalent, derivative, variant, mutant or fragment of Transglutaminase 2. Functional in this context means having a function of Transglutaminase 2 that is involved in its biological activity according to the invention.

The biological activity of Stearyl-CoA-Desaturase (SEQ ID NO. 11,12) according to the present invention, i. e. mediating the involvement of a macrophage in an inflammatory process according to the invention, is dependent, for example, on binding to a substrate, e. g. palmitoyl-CoA and/or stearyl-CoA and/or on its oxidative activity and/or on any other Stearyl-CoA-Desaturase function, e. g. substrate recognition, relevant for its biological activity according to the invention.

The invention also concerns a functional equivalent, derivative, variant, mutant or fragment of Stearyl-CoA-Desaturase. Functional in this context means having a function of Stearyl-CoA-Desaturase that is involved in its biological activity according to the invention.

The biological activity of UDP-Glucose Ceramide Glycosyltransferase (SEQ ID NO.

13,14) according to the present invention, i. e. mediating the involvement of a macrophage in an inflammatory process according to the invention, is dependent, for example, on binding to a substrate, e. g. UDP-glucose and/or ceramide and/or on its transferring activity and/or on any other UDP-Glucose Ceramide Glycosyltransferase function, e. g. substrate recognition, relevant for its biological activity according to the invention.

The invention also concerns a functional equivalent, derivative, variant, mutant or fragment of UDP-Glucose Ceramide Glycosyltransferase. Functional in this context means having a function of UDP-Glucose Ceramide Glycosyltransferase that is involved in its biological activity according to the invention.

According to the present invention, the biological activity of a protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA- Desaturase and UDP-Glucose Ceramide Glycosyltransferase, if expressed at a lower level than the control level, is preferably activated in order to inhibit hyperactivation or reduce a hyperactivated state of a macrophage, and if expressed at a higher level than the control level, is preferably inhibited in order to inhibit hyperactivation or reduce a hyperactivated state of a macrophage.

In one embodiment the present invention concerns a test method for determining a substance to be an activator or inhibitor of protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP- Glucose Ceramide Glycosyltransferase. Since such a protein is involved in a chronic inflammatory airway disease and plays a role in mediating inflammation, a substance modulating the biological activity of such a protein can be used for treating a chronic

inflammatory airway diseases or can be used as lead compound for optimization of the function of the substance in a way that the optimized substance is suitable for treating chronic inflammatory airway diseases. For performing a method of the invention, a test system according to the invention can be used.

The present invention also concerns a test system for determining whether a substance is an activator or an inhibitor of a protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase. A test system useful for performing a method of the invention comprises a cellular or a cell-free system. For example, one embodiment of to the invention concerns a test system that is designed in a way to allow the testing of substances acting on the expression level of the differentially expressed nucleic acid sequence e. g. using expression of a reporter-gene, e. g. luciferase gene or the like, as a measurable readout. Another embodiment of the invention concerns a test system that is designed in a way to allow the testing of substances directly interacting with a respective function of a protein of the invention or interfering with the respective activation of a function a protein of the invention by a natural or an artificial but appropriate activator of the respective protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase, e. g. an appropriate kinase or the like.

A test system according to the invention comprises a protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase, or a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention, a nucleic acid encoding a said protein or encoding a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention and/or regulatory elements, wherein a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention or a nucleic acid encoding a said protein or a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention is capable to interact with a substance which should be tested in a way that direct interaction leads to a measurable read-out indicative for the change of a respective

biological activity of a said protein according to the invention and/or for the change of expression of a said protein of the invention.

A test system of the invention comprises, for example, elements well known in the art. For example, cell-free systems may include, for example, a said protein or a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention, a nucleic acid encoding a said protein or encoding a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention in soluble or bound form or in cellular compartments or vesicles. Suitable cellular systems include, for example, a suitable prokaryotic cell or eukaryotic cell, e. g. such comprising a said protein of the invention or a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention, a nucleic acid encoding a said protein or encoding a functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention. A cell suitable for use in a said test system of the invention may be obtained by recombinant techniques, e. g. after transformation or transfection with a recombinant vector suitable for expression of a desired protein of the invention or functional equivalent, derivative, variant, mutant or fragment of a said protein of the invention, or may e. g. be a cell line or a cell isolated from a natural source expressing a desired protein of the invention or functional equivalent, derivative, variant, mutant or fragment of a said protein. A test system of the invention may include a natural or artificial ligand of the protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP- Glucose Ceramide Glycosyltransferase if desirable or necessary for testing whether a substance of interest is an inhibitor or activator of a said protein of the invention.

A test method according to the invention comprises measuring a read-out, e. g. a phenotypic change in the test system, for example, if a cellular system is used a phenotypic change of the cell. Such change may be a change in a naturally occurring or artificial response, e. g. a reporter gene expression of the cell to a protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase activation or inhibition, e. g. as detailed in the Examples hereinbelow.

A test method according to the invention can on the one hand be useful for high throughput testing suitable for determining whether a substance is an inhibitor or activator of the invention, but also e. g. for secondary testing or validation of a hit or lead substance identified in high throughput testing.

The present invention also concerns a substance identified in a method according to the invention to be an inhibitor or activator of a protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase. A substance of the present invention is any compound which is capable of activating or preferably inhibiting a function of a protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase. An example of a way to activate or inhibit a function of a protein selected from the group consisting of : MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase is by influencing the expression level of a said protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP- Glucose Ceramide Glycosyltransferase. Another example of a way to activate or inhibit a function of a protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase is to apply a substance directly binding a protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase and thereby activating or blocking functional domains of a said protein of the invention, which can be done reversibly or irreversibly, depending on the nature of the substance applied.

Accordingly, a substance useful for activating or inhibiting biological activity of a protein selected from the group consisting of MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase includes a substance acting on the expression of differentially expressed nucleic acid sequence, for example a nucleic acid fragment hybridizing with the corresponding gene or regulator sequence and thereby influencing gene

expression, or a substance acting on a protein selected from the group consisting of: MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP- Glucose Ceramide Glycosyltransferase itself or on its activation or inhibition by other naturally, occurring cellular components, e. g. an other protein acting enzymatically on a said protein of the invention, e. g. a protein kinase.

Therefore, the invention concerns, for example, a substance which is a nucleic acid sequence coding for the gene of a protein of the invention, or a fragment, derivative, mutant or variant of such a nucleic acid sequence, which nucleic acid sequence or a fragment, derivative, mutant or variant thereof is capable of influencing the gene expression level, e. g. a nucleic acid molecule suitable as antisense nucleic acid, ribozyme, or for triple helix formation.

The invention also concerns a substance which is e. g. an antibody or an organic or inorganic compound directly binding to or interfering with the activation of a protein selected from the group consisting of : MIF, DAD1, ARL4, GNS, Transglutaminase 2, Stearyl-CoA-Desaturase and UDP-Glucose Ceramide Glycosyltransferase and thereby affecting its biological activity.

In a further aspect, the present invention relates to a method for determining an expression level of a nucleic acid coding for a protein of the invention, preferably messenger RNA, or protein of the invention itself, in cell, preferably in a macrophage, more preferably in a macrophage isolated form a site of inflammation, even more preferably from a site of inflammation in a subject suffering from a chronic inflammatory airway disease. Such a method can be used, for example, for testing whether a substance is capable of influencing differentially expressed nucleic acid sequence expression levels in a method outlined above for determining whether a substance is an activator or inhibitor according to the present invention. A method for determining an expression level according to the invention can, however, also be used for testing the activation state of a macrophage, e. g. for diagnostic purposes or for investigation of the success of treatment for a disease which is caused by the hyperactivated macrophage. Said macrophage is preferably a mammalian, more preferably a human cell. Accordingly, macrophages of the present invention are

preferably obtainable from the site of inflammation in a mammal and more preferably from a site of inflammation in a human being. Accordingly, the invention also relates to a method for diagnosis of a chronic inflammatory disease, or monitoring of such disease, e. g. monitoring success in treating beings in need of treatment for such disease, comprising determining an expression level of a nucleic acid coding for a protein of the invention, preferably messenger RNA, or protein of the invention itself in a macrophage.

A method for determining expression levels of a nucleic acid coding for a protein of the invention, preferably messenger RNA, or protein of the invention itself can, depending on the purpose of determining the expression level, be performed by known procedures such as measuring the concentration of respective RNA transcripts via hybridization techniques or via reporter gene driven assays such as luciferase assays or by measuring the protein concentration of said protein of the invention using respective antibodies.

The present invention also relates to the use of a substance according to the invention for the treatment for a chronic inflammatory airway disease. Another embodiment of the present invention relates to a pharmaceutical composition comprising at least one of the substances according to the invention determined to be an activator or an inhibitor. The composition may be manufactured in a manner that is itself known, e. g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, powdering, emulsifying, encapsulating, entrapping or lyophilizing processes.

In order to use substances activating or inhibiting according to the invention as drugs for treatment for chronic inflammatory airway diseases, the substances can be tested in animal models for example an animal suffering from an inflammatory airway disorder or a transgenic animal expressing protein of the invention.

Toxicity and therapeutic efficacy of a substance according to the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LDSO and ED50. The data

obtained are used for estimating the animal or more preferred the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays ampules, etc.) and the administration route (for example transdermal, oral, buccal, nasal, enteral, parenteral, inhalative, intratracheal, or rectal).

A pharmaceutical composition containing at least one substance according to the invention as an active ingredient can be formulated in conventional manner. Methods for making such formulations can be found in manuals, e. g."Remington Pharmaceutical Science". Examples for ingredients that are useful for formulating at least one substance according to the present invention are also found in WO 99/18193, which is hereby incorporated by reference.

In a further aspect the invention concerns a method for treating a chronic inflammatory airway disease. Such method comprises administering to a being, preferably to a human being, in need of such treatment a suitable amount of a pharmaceutical composition comprising at least one substance determined to be an activator or inhibitor by a method according to the invention.

In an other embodiment the invention relates to a method for selectively modulating the concentration of a protein of the invention in a macrophage, comprising administering a substance determined to be an activator or inhibitor of protein of the invention.

The following examples are meant to illustrate the present invention, however, shall not be construed as limitation. However, the Examples describe most preferred embodiments of the invention.

Examples Example 1: Comparative Expression Profilinq

The following is an illustration of how comparative expression profiling can be performed in order to identify protein of the invention.

1. 1. Selection of Subjects Three groups of subjects are studied: healthy non-smokers, healthy smokers and patients with COPD.

In order to assess lung function subjects have to perform spirometry. A simple calculation based on age and height is used to characterize the results. COPD subjects are included if their FEV, % predicted is <70%. Healthy smokers are age and smoking history matched with the COPD subjects but have normal lung function.

Healthy non-smokers have normal lung function and have never smoked. The latter group has a methacholine challenge to exclude asthma. This technique requires increasing doses of methacholine to be given to the subject, with spirometry between each dose. When the FEV, falls 20% the test is stopped and the PCZO is calculated.

This is the dose of methacholine causing a 20% fall in FEV, and we will require a value of >32 as evidence of absence of asthma. All subjects have skin prick tests to common allergens and are required to have negative results. This excludes atopic individuals. The clinical history of the subjects is monitored and examined in order to exclude concomitant disease.

1.2. BAL (bronchoalveolar lavage) Procedure Subjects are sedated with midazolam prior to the BAL. Local anaesthetic spray is used to anaesthetize the back of the throat. A 7mm Olympus bronchoscope is used.

The lavaged area is the right middle lobe. 250 ml of sterile saline is instiled and immediately aspirated. The resulting aspirate contains macrophages.

1.3. BAL Processing BAL is filtered through sterile gauze to remove debris. The cells are washed twice in HBSS, resuspended in 1 ml HBSS (Hank's Balanced Salt Solution) and counted. The macrophages are spun to a pellet using 15 ml Falcon blue-cap polypropylen, resuspended in Trizol reagent (Gibco BRL Life Technologies) at a concentration of 1 ml Trizol reagent per 10 million cells and then frozen at-70°C.

1.4. Differential Gene Expression Analysis Total RNA is extracted from macrophage samples obtained according to Example 1.3. Cell suspensions in Trizol are homogenized through pipetting and incubated at room temperature for 5 minutes. 200 lli chloroform per ml Trizol is added, the mixture carefully mixed for 15 seconds and incubated for 3 more minutes at room temperature. The samples are spun at 10000g for 15 minutes at 4°C. The upper phase is transferred into a new reaction tube and the RNA is precipitated by adding 0.5 ml isopropanol per ml Trizol for 10 minutes at room temperature. Then, the precipitate is pelleted by using a microcentifuge for 10 minutes at 4°C with 10000g, the pellet is washed twice with 75% ethanol, air dried and resuspended in DEPC- H2O.

An RNA cleanup with Qiagen RNeasy Total RNA isolation kit (Qiagen) is performed in order to improve the purity of the RNA. The purity of the RNA is determined by agarose gelelectrophoresis and the concentration is measured by UV absorption at 260 nm.

5 llg of each RNA is used for cDNA synthesis. First and second strand synthesis are performed with the SuperScript Choice system (Gibco BRL Life Technologies). In a total volume of 11 lli RNA and 1 pl of 100 1M T7-(dt) 24 primer, sequence shown in SEQ ID NO. 15, are heated up to 70°C for 10 minutes and then cooled down on ice for 2 minutes. First strand buffer to a final concentration of 1x, DTT to a concentration of 10 mM and a dNTP mix to a final concentration of 0.5 mM are added to a total volume of 18 pI. The reaction mix is incubated at 42°C for 2 minutes and 2 ; n. i of Superscript it reverse transcriptase (200 U/lli) are added. For second strand synthesis 130 ii of a mix containing 1.15x second strand buffer, 230 p. M dNTPs, 10 U E. coli DNA ligase (10U/µl), E. coli DNA polymerase (10 U/µl), RNase H (2U/µl) is added to the reaction of the first strand synthesis and carefully mixed with a pipette. Second strand synthesis is performed at 16°C for 2 hours, then 2 µl of T4 DNA polymerase (5 U/µl) are added, incubated for 5 minutes at 16°C and the reaction is stopped by adding 10 lli 0.5 M EDTA.

Prior to cRNA synthesis the double stranded cDNA is purified. The cDNA is mixed with an equal volume of phenol : chloroform : isoamylalcohol (25: 24: 1) and spun through the gel matrix of phase lock gels (Eppendorf) in a microcentrifuge in order to separate the cDNA from unbound nucleotides. The aqueous phase is precipitated with ammoniumacetate and ethanol. Subsequently, the cDNA is used for in vitro transcription. cRNA synthesis is performed with the ENZO BioArray High Yield RNA Transcript Labeling Kit according to manufacturer's protocol (ENZO Diagnostics).

Briefly, the cDNA is incubated with 1x HY reaction buffer, 1x biotin labeled ribonucleotides, 1x DTT, 1x RNase Inhibitor Mix and 1x T7 RNA Polymerase in a total volume of 40 lli for 5 hours at 37°C. Then, the reaction mix is purified via RNeasy columns (Qiagen), the cRNA precipitated with ammonium acetate and ethanol and finally resuspended in DEPC-treated water. The concentration is determined via UV spectrometry at 260 nm. The remaining cRNA is incubated with 1x fragmentation buffer (5x fragmentation buffer: 200 mM Tris acetate, pH 8.1,500 mM KOAc, 150 mM MgOAc) at 94°C for 35 minutes.

For hybridization of the DNA chip 15 jug of cRNA is used, mixed with 50 pM biotin- labeled control B2 oligonucleotide, sequence shown SEQ ID NO. 16,1x cRNA cocktail, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 1x MES (2- [N- morpholino]-ethanesulfonic acid) hybridization buffer in a total volume of 300} I. The hybridization mixture is heated up to 99°C for 5 minutes, cooled down to 45°C for 10 minutes and 200 lli of the mix are used to fill the probe array. The hybridization is performed at 45°C at 60 rpm for 16 hours.

After the hybridization the hybridization mix on the chip is replaced by 300 lli non- stringent wash buffer (100 mM MES, 100 mM NaCI, 0.01% Tween 20). The chip is inserted into an Affymetrix Fluidics station and washing and staining is performed according to the EukGE-WS2 protocol. The staining solution per chip consists of 600 pl 1x stain buffer (100 mM MES, 1 M NaCI, 0.05% Tween 20), 2 mg/ml BSA, 10 pLg/ml SAPE (streptavidin phycoerythrin) (Dianova), the antibody solution consists of 1x stain buffer, 2 mg/mi BSA, 0.1 mg/m1 goat gG, 3, ug/ml biotinylated antibody.

After the washing and staining procedure the chips are scanned on the HP Gene Array Scanner (Hewlett Packard).

Data Analysis is performed by pairwise comparisons between chips hybridized with RNA isolated from COPD smokers and chips hybridized with RNA isolated from healthy smokers.

The following is an illustration of differentially expressed genes and their function as identified according to the approach of the present invention.

Example 2 : MIF A gene identified as consistently upregulated in individuals with COPD codes for MIF. MIF is secreted by pituitary cells, macrophages, and T cells and its synthesis can be induced by proinflammatory stimuli such as LPS, TNFa, and IFN-y. MIF itself has proinflammatory activity by counteracting suppressive effects of glucocorticoids and by inducing inflammation in response to invasion of bacteria. Neutralizing MIF can prevent septic shock in certain mouse models (Calandra et al. 1994, Bernhagen et al. 1998, Calandra et al. 2000) MIF is consistently found upregulated (42%) in COPD smokers compared to healthy smokers. This is shown by"fold change"values (Tab. 1). The p value for comparing COPD smokers and healthy smokers is 0.03.

Tab. 1 : Deregulation of MIF :"fold change" (FC) values for each patient are listed for the comparisons between obstructed and healthy smokers. comp FC comp FC comp FC comp FC 1 vs 2-1. 3 5 vs 43 3.9 39 vs 57-2.0 68 vs 66 2. 8 1 vs 37 8. 0 5 vs 56 1. 9 39 vs 58 1. 0 68 vs 69 2. 3 1 vs 43 1. 8 5 vs 57 1.5 39 vs 62 1. 0 68 vs 76 5. 0 1 vs 56-1. 3 5 vs 58 2. 9 44 vs 2 1. 4 68 vs 78 3. 2 1 vs 57-1. 6 5 vs 62 2.0 44 vs 37 14.4 70 vs 65 1. 1 1 vs 58 1. 2 6 vs 2-1. 6 44 vs 43 3. 0 70 vs 66 1. 4 1 vs 62-1. 2 6 vs 37 6. 5 44 vs 56 1. 4 70 vs 69 1. 1 3 vs 2-1. 6 6 vs 43 1.5 44 vs 57 1. 1 70 vs 76 2. 6 3 vs 37 6. 3 6 vs 56-1. 6 44 vs 58 2. 1 70 vs 78 1. 6 3 vs 43 1.4 6 vs 57-2. 0 44 vs 62 1.5 71 vs 65 2.1 3 vs 56-1. 6 6 vs 58 1. 0 64 vs 65 2. 0 71 vs 66 2.7 3 vs 57-2. 1 6 vs 62-1. 5 64 vs 66 2. 6 71 vs 69 2. 2 3 vs 58-1. 1 39 vs 2-1. 6 64 vs 69 2.1 71 vs 76 4.9 3 vs 62-1. 5 39 vs 37 1.0 64 vs 76 4.7 71 vs 78 3. 1 5 vs 2 1. 9 39 vs 43 1. 0 64 vs 78 3.0 5 vs 37 18.5 39 vs 56-1. 5 68 vs 65 2.1

2.1. Cloning of MIF MIF is cloned from a total RNA extracted from human THP-1 cells. 5 tg RNA is reverse transcribed into cDNA with 5 ng oligo (dt) 18 primer, 1x first strand buffer, 10 mM DTT, 0.5 mM dNTPs and 2 U Superscript II (Gibco BRL) at 42°C for 50 minutes.

Then, the reaction is terminated at 70°C for 15 minutes and the cDNA concentration is determined by UV-spectrophotometry. For amplification of MIF 100 ng of the cDNA and 10 pmoles of sequence-specific primers for MIF (forward primer, SEQ ID NO. 17 and reverse primer, SEQ ID NO. 18) are used for PCR. Reaction conditions are: 2 minutes of 94°C, 35 cycles with 30 seconds at 94°C, 30 seconds at 53°C, 90 seconds at 72°C, followed by 7 minutes at 72°C with Taq DNA-polymerase. The reaction mix is separated on a 2% agarose gel, a band of about 360bp is cut out and purified with the QIAEX 11 extraction kit (Qiagen). The concentration of the purified band is determined and about 120 ng are incubated with 300 ng of pDONR201, the donor vector of the Gateway system (Life Technologies), 1x BP clonase reaction buffer, BP clonase enzyme mix in a total volume of 20 ul for 60 minutes at 25°C.

Then, reactions are incubated with 2 lli of proteinase K and incubated for 10 minutes at 37°C. The reaction mix is then electroporated into competent DB3.1 cells and plated on Kanamycin-containing plates. Clones are verified by sequencing. A clone, designated pDONR-MIF, with identical sequence to the database entry (acc.

L19686) is used for further experiments.

2.2. Generation of a transfection vector for MIF

The vector containing MIF described under 1.1. is used to transfer the cDNA for MIF to the expression vector pcDNA3.1 (+)/attR that contains the"attR1"and"attR2" recombination sites of the Gateway cloning system (Life Technologies) where MIF is expressed under the control of the CMV promoter. 150 ng of the"entry vector" pDONR-MIF is mixed with 150 ng of the"destination vector"pcDNA3.1 (+)/attR, 4 ptl of the LR Clonase enzyme mix, 4 pI LR Clonase reaction buffer, added up with TE (Tris/EDTA) to 20 µl and incubated at 25°C for 60 minutes. Then, 2 µl of proteinase K solution is added and incubated for 10 minutes at 37°C. 1 lli of the reaction mix is transformed into 50 DH5a by a heat-shock of 30 seconds at 42°C after incubating cells with DNA for 30 minutes on ice. After heat-shock of the cells 450 µl of S. O. C. is added and cells are incubated at 37°C for 60 minutes. Cells (100 aI) are plated on LB plates containing 100 lig/ml ampicillin and incubated over night.

A colony that contains pcDNA3.1 (+)/attR with MIF as an insert is designated pcDNA/MIF and used for transfection studies.

2.3. Expression of recombinant MIF The vector containing MIF described under 1.1. is used to transfer the cDNA for MIF to the expression vectors gpET28abc/attR that contains the"attR1"and"attR2" recombination sites of the Gateway cloning system (Life Technologies). These vectors allow the expression of recombinant hig-tagged MIF in bacteria under the control tog the T7 promoter. 150 ng of the"entry vector"pDONR-MIF is mixed with 150 ng of the"destination vector"gpET28abc/attR, 4 lli of the LR Clonase enzyme mix, 4 lli LR Clonase reaction buffer, added up with TE (Tris/EDTA) to 20 lli and incubated at 25°C for 60 minutes. Then, 2 lli of proteinase K solution is added and incubated for 10 minutes at 37°C. 1 pLi of the reaction mix is transformed into 50 gel DH5a by a heat-shock of 30 seconds at 42°C after incubating cells with DNA for 30 minutes on ice. After heat-shock of the cells 450 ill of S. O. C. is added and cells are incubated at 37°C for 60 minutes. Cells (100 aI) are plated on LB plates containing 100 lig/ml ampicillin and incubated over night.

A colony that contains gpET28abc/attR with MIF fused to the his-tag in the correct reading frame is designated pgPET/MIF and used for expression of MIF in bacteria.

2.4. Purification of recombinant MIF 1 I LB broth including 100 ug/ml ampicillin is inoculated with 0.5 ml of an overnight culture of E. coli M15 (pREP4) that carries pQE/MIF. The culture is incubated at 37°C with vigorous shaking until OD6oo of 0.6. Expression is induced by adding 1 mM IPTG and the culture is grown further for 4 hours. Cells are harvested by centrifugation at 4000xg for 20 minutes at 4°C. Pellet is frozen at-20°C.

Cells are thawed on ice and resuspended in 2 mf/g cell pellet of lysis buffer (50 mM NaH2P04, pH 8.0,300 mM NaCl, 10 mM imidazole). Then, lysozyme is added to 1 mg/ml and incubated on ice for 30 minutes. Then, cells are sonicated (six bursts of 10 seconds at 300 W). 10 lig/ml RNase A and 5 lig/ml DNase I is added and incubated on ice for 10 minutes. Then, lysates are cleared by spinning debris at 10000xg for 20 minutes at 4°C. Then, protease inhibitors (40 pg/ml bacitracin, 4 Ag/ml leupeptin, 4 Hg/ml chymostatin, 10 pg/ml pefabloc, 100 juM PMSF) are added.

3 ml of Ni-NTA resin (Qiagen) are added to the lysate and filled into a column.

Binding to the resin is allowed for 60 minutes at 4°C during gentle shaking. Then, column outlet is opened, the resin washed twice with 12 mi wash buffer (50 mM NaH2PO4, pH 8.0,300 mM NaCl, 20 mM imidazole) and eluted with four times 3 ml of elution buffer (50 mM NaH2PO4, pH 8.0,300 mM NaCl, 250 mM imidazole). The elution fraction that contains the recombinant protein is determined by SDS-PAGE and protein concentration of the purified protein is determined by the method of Bradford.

2.5. Purification of CD4+ T cells and mononuclear cells from periperal blood 10 ml blood of healthy volunteers is diluted with 25 ml PBS and layered carefully on top of 15 ml ficoll in a 50 ml Falcon tube. The tube is spun at 400x g for 40 minutes at room temperature. Cells are removed with a pasteur pipet and washed in 50 ml PBS at 500x g for 10 minutes at RT.

CD4+ lymphocytes are isolated with the help of magnetic beads. The cell fraction (as described in the previous paragraph) is resuspended 80 ul MACS buffer (PBS, 2 mM EDTA, 0.5% BSA) per 1 cells. 20 pl of CD4+ separation beads (Miltenyi Biotech) are added to 1 cells, mixed and incubated at 4°C for 15 minutes. Then, 20 volumes of MACS buffer are added and spun at 1000 rpm for 10 minutes. The pellet

is resuspended in 500 ul MACS buffer per 1X108 cells and added to a Miltenyi Separation Column LS+ that is equilibrated with 3 ml of MACS buffer. Magnetic beads are exposed to a magnetic field for 30 seconds and labeled CD4+ cells are retained. Afterwards, the columns is separated from the magnetic field and CD4+ cells are flushed out with 5 ml of MACS buffer. Cells are spun down and resuspended in RPM11640, 10% FCS).

Similarly, human mononuclear cells are isolated from whole blood by ficoll density centrifugation. After seeding the cells are washed twice in 24 hours with RPMI 1640, 10% FCS in order to remove non-adherent cells.

2.6. Phenotypic/cellular effects caused by MIF The following assays are performed with cell lines THP-1 (Tsuchiya et al. 1980), and MonoMac 6 (Ziegler-Heitbrock et al. 1988) that are transiently or stably transfected with MIF and the read-outs are compared to mock-transfected cells. In addition substances according to the invention that stimulate the activity of MIF are added.

Production and Release of Cytokines Monocytic/macrophage cell lines are stimulated with MIF (1 Lg/ml) at cell densities between 2.5 and 5 x 105 cells/ml. Cells are harvested after 0,1,3,6,12,24,48, and 72 hours, the supernatant frozen for further investigation, cells are washed with PBS, and resuspended in 400 ; J of RLT buffer (from Qiagen RNeasy Total RNA Isolation Kit) with 143 mM ß-mercaptoethanol, the DNA sheared with a 20 g needle for at least 5 times and stored at-70°C.

Stimulation of cells by cigarette smoke is performed by a smoke-enriched media. 100 ml RPMI media without supplements is perfused with the cigarette smoke of 2 cigarettes. The smoke of the cigarettes is pulled into a 50 ml syringe (about 20 volumes of a 50-ml volumes per cigarette) and then perfused into the media.

Afterwards, the pH of the media is adjusted to 7.4, and the media is filtersterilized through a 0.211m filter. Cells are resuspended in smoke-enriched media and incubated for 10 minutes at 37°C at a density of 1X106 cells/ml. Then, cells are washed twice with RPMI 1640 and seeded in flasks or 24-well plates. (MonoMac6) for the times indicated above.

Total RNAs are isolated with the Qiagen RNeasy Total RNA Isolation Kit (Qiagen) according to the manufacturer's protocol. Purified RNA is used for TaqMan analysis.

The expression levels of cytokines TNFa, IL-1p, IL-8, and IL-6 are measured.

Detection of secreted cytokines Proteins in the supernatants of the cultured and stimulated cells are precipitated by adding TCA to a final concentration of 10%. Precipitates are washed twice with 80% ethanol and pellets are resuspended in 50 mM Tris/HCI, pH 7.4,10 mM MgCI2, 1 mM EDTA. Protein concentration is determined via the Bradford method and 50 Dg of each sample are loaded on 12% SDS polyacrylamide gels. Gels are blotted onto PVDF-membranes, blocked for 1 hour in 5% BSA in TBST, and incubated for 1 hour with commercially available antibodies against human TNFa, IL-1, IL-8, and IL-6.

After washing with TBST blots are incubated with anti-human IgG conjugated to horseradish-peroxidase, washed again and developed with ECL chemiluminescence kit (Amersham). Intensity of the bands are visualised with BioMax X-ray films (Kodak) and quantified by densitometry.

Purified CD4+ cells (as described under 2.0) are seeded in 96-well-plates (5x104 cells/200, ul) in RPMI 1640,10% FCS and incubated with dexamethasone (10 nM) in the presence or absence of 10 ng/ml MIF. After 24 hours of incubation at 37°C in a humidified atmosphere with 5% CO2, cytokine release (e. g. IL-2 or IFN-y (interferon- gamma)) is determined by ELISA. MIF overrides the inhibitory effect of dexamethasone and causes release of cytokines. The counteracting effect of MIF on dexamethasone is modulated by adding substances according to the invention (0.1- 100 ng/ml) to the reaction mix and calculate the effect as percent inhibition of the MlF-mediated effect.

In order to determine cytokine release (IL-1 ß, IL-6, IL-8, TNF-o) in monocytes, the cells need to be treated with 1 pg/ml LPS after 1 hour of preincubation with dexamethasone and MIF (according to previous paragraph).

Detection of secreted matrix metalloproteases and other proteases

The procedure is identical to the one used for cytokines. Antibodies used for Western blotting are against human MMP-1, MMP-7, MMP-9, and MMP-12.

Activity of secreted matrix metalloproteases Protease activity is determined with a fluorescent substrate. Supernatants isolated from stimulated and unstimulated cells (described above) are incubated in a total volume of 50 pI with 1 wu of the substrate (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu (EDANS)-Ala-Lys-NH2 (Novabiochem)) for 5 minutes at room temperature. Positive controls are performed with 125 ng purified MMP-12 per reaction. Protease activity is determined by fluorometry with an excitation at 320 nm and an emission at 405 nm.

In an alternative assay to determine proteolytic activity and cell migration a chemotaxis (Boyden) chamber is used. In the wells of the upper part of the chamber cells (105 cells per well) are plated on filters coated with an 8 um layer of Matrigel (Becton Dickinson). In the lower compartment chemoattractants like MIF (1 gg/ml), leukotriene B4 (10 ng/ml), MCP-1 (10 ng/ml) are added to the media. After five days filters are removed, cells on the undersurface that have traversed the Matrigel are fixed with methanol, stained with the Diff-Quik staining kit (Dade Behring) and counted in three high power fields (400x) by light microscopy.

Chemotaxis Assay In order to determine chemotaxis, a 48 well chemotaxis (Boyden) chamber (Neuroprobe) is used. Cells are starved for 24 hours in RPMI media without FCS.

Chemotaxis is stimulated by 100 ng/ml LPS, 10 ng/ml leukotriene B4, or MCP-1.

Additon of MIF (1 wg/ml) is used to block chemotaxis. Substances according to the invention are diluted in RPMI media without FCS and 30 ; J is placed in the wells of the lower compartment in order to counteract MIF activity. The upper compartment is separated from the lower compartment by a polycarbonate filter (pore size 8 pLm). 50 jj ce) ! suspension (5 x104) are placed in the well of the upper compartment. The chamber is incubated for 5 hours at 37°C in a humidified atmosphere with 5% CO2' Then the filter is removed, cells on the upper side are scraped off, cells on the downside are fixed for 5 minutes in methanol and stained with the Diff-Quik staining

set (Dade Behring). Migrated cells are counted in three high-power fields (400x) by light microscopy.

Adherence Assay Cells are harvested, washed in PBS and resuspended (4x106/ml) in PBS and 1 gM BCECF ((2'-7'-bis-(carboxyethyl)-5 (6')-carboxyfluorescein acetoxymethyl) ester, Calbiochem) and incubated for 20 minutes at 37°C. Cells are washed in PBS and resuspended (3.3x 106/ml) in PBS containing 0.1% BSA. 3x105 cells (90, vLI) are added to each well of a 96-well flat bottom plate coated with laminin (Becton Dickinson) and allowed to settle for 10 minutes. Substances according to the invention are added in the presence and absence of MIF (1 ug/ml), and plates are incubated for 20 minutes at 37°C. Cells are washed with PBS containing 0.1% BSA and adherent cells are solubilized with 100 jd of 0.025 M NaOH and 0.1% SDS.

Quantification is performed by fluorescence measurement.

Phagocytosis Cell suspensions (2.5x104 cells/ml) are seeded in 6-well plates with 5 ml of U937 or THP-1 or in 24-well plates with 2 mi of MonoMac6 and incubated for 1 hour at 37°C in a humidified atmosphere with 5% CO2. In the presence of MIF, substances according to the invention are added to counteract the actvity of MIF. 40 pl of a dispersed suspension of heat-inactivated Saccharomyces boulardii (20 yeast/cell) are added to each well. Cells are incubated for three more hours, washed twice with PBS and cytocentrifuged. The cytospin preparations are stained with May-Grunwald- Giemsa and phagocytosed particles are counted by light microsopy.

Example 3: DAD1 A gene identified as being downregulated in COPD smokers compared to healthy smokers is DAD1 (defender against apoptotic cell death 1). Originally, DAD1 was discovered as being a negative regulator of apoptosis (Nakashima et al. 1993). By homology to the Ost2 protein in Schizosaccaromyces pombe it was identified as the 16 kDa subunit of the oligosaccaryltransferase complex which catalyzes the transfer

of high mannose oligosaccharides onto asparagine residues in nascent polypeptides.

DAD1 is an integral membrane protein and is ubiquitously expressed (Kelleher and Gilmore 1997).

DAD1 is consistently found upregulated (42%) in comparisons between COPD smokers and healthy smokers. This is shown by"fold change"values (Tab. 2).

Tab. 2: Fold change values (FC) for comparisons between obstructed smokers and healthy smokers On average DAD1 is upregulated by 1.6fold, the median is 1.5fold. comp FC comp FC comp FC comp FC 1 vs 2 -1.1 5 vs 43 2.3 39 vs 57 4.8 68 vs 66 1.4 1 vs 37 2.5 5 vs 56 3.9 39 vs 58 2.5 68 vs 69 1. 1 vs 43 1.5 5 vs 57 4.0 39 vs 62 6.6 68 vs 76 2. 2 1 vs 56 2.4 5 vs 58 2.0 44 vs 2 -2.9 68 vs 78 2.1 1 vs 57 2.5 5 vs 62 5.5 44 vs 37 1.1 70 vs 65 -1. 3 1 vs 58 1.3 6 vs 2 1.0 44 vs 43 -1.7 70 vs 66 -1.4 1 vs 62 34 6 vs 37 27 44 vs 56 1 0 70 vs 69-1 3 3 vs 2-1. 2 6 vs 43 1. 6 44 vs 57 1. 0 70 vs 76 1. 1 3 vs 37 2. 3 6 vs 56 2. 7 44 vs 58-19 70 vs 78 11 3 vs 43 1.4 6 vs 57 2.76 44 vs 62 1.4 71 vs 65 1.1 3 vs 56 23 6 vs 58 14 64 vs 65-1z1 71 vs 66 1 0 3 vs 57 2.3 6 vs 62 3.7 64 vs 66 -1.1 71 vs 69 1.2 3 vs 58 1.2 39 vs 2 1.7 64 vs 69 -1.1 71 vs 76 1. 6 3 vs 62 3.2 39 vs 37 4.8 64 vs 76 1.3 71 vs 78 1.6 5 vs 2 1.4 39 vs 43 2.8 64 vs 78 1. 3 5 vs 37 3.9 39 vs 56 4.7 68 vs 65 1.4 The protein is cloned and assays are designed and performed in an analogous manner to the cloning and assays described hereinbefore.

Example 4: ARL4,

A gene identified as being upregulated in COPD smokers compared to healthy smokers is ARL4 (ADP-ribosylation factor-like protein 4). ARLs belong to the family of ADP-ribosylation factors (ARFs). ARFs are involved in vesicular and membrane trafficking. ARL4 is both detected inside and outside of the nucleus and it is speculated that it is involved in cellular differentiation (Jacobs et al. 1999).

ARL4 is consistently found upregulated (45%) in comparisons between COPD smokers and healthy smokers. This is shown by"fold change"values (Tab. 3: The p values for two separate groups comparing COPD smokers and healthy smokers are 0.10 and 0.06.

Tab. 3: Fold change values (FC) for comparisons between obstructed smoker and healthy smokers. On average ARL4 is upregulated by 1.6fold, the median is 1.9fold. comp FC comp FC comp FC comp FC 1 vs 2-1. 1 5 vs 43 1.9 39 vs 57 2.5 68 vs 66 2.4 1 vs 37 2.7 5 vs 56 2.2 39 vs 58 1.2 68 vs 69 4.5 1 vs 43 3. 2 5 vs 57 1.6 39 vs 62 1. 5 68 vs 76 7.8 1 vs 56 4.3 5 vs 58-1. 2 44 vs 2-3. 7 68 vs 78 3. 3 1 vs 57 2. 0 5 vs 62 1. 0 44 vs 37-1. 3 70 vs 65 1.2 1 vs 58-1. 1 6 vs 2 1. 2 44 vs 43-1. 1 70 vs 66 1. 5 1 vs 62 1. 2 6 vs 37 3. 4 44 vs 56 1. 5 70 vs 69 2. 7 3 vs 2-1. 8 6 vs 43 3. 6 44 vs 57-1. 7 70 vs 76 4.7 3 vs 37 2.0 6 vs 56 4. 1 44 vs 58-3. 5 70 vs 78 1.9 3 vs 43 2. 4 6 vs 57 2. 7 44 vs 62-2. 7 71 vs 65 1.7 3 vs 56 3. 2 6 vs 58 1. 3 64 vs 65-1. 1 71 vs 66 2.0 3 vs 57 1.5 6 vs 62 1.6 64 vs 66 1.2 71 vs 69 3. 9 3 vs 58 -1.4 39 vs 2 1.1 64 vs 69 2.2 71 vs 76 6.7 3 vs 62 1. 0 39 vs 37 3. 3 64 vs 76 3.8 71 vs 78 2.8 5 vs 2-1. 3 39 vs 43 4. 0 64 vs 78 1.6 5 vs 37 1.8 39 vs 56 4.7 68 vs 65 1.9

4.1. Cloning of ARL4 ARL4 is cloned from a total RNA extracted from human 3T3-L1. 5 jug RNA is reverse transcribed into cDNA with 5 ng oligo (dt) 18 primer, 1x first strand buffer, 10 mM DTT, 0.5 mM dNTPs and 2 U Superscript II (Gibco BRL) at 42°C for 50 minutes. Then, the reaction is terminated at 70°C for 15 minutes and the cDNA concentration is determined by UV-spectrophotometry. For amplification of ARL4100 ng of the cDNA and 10 pmoles of sequence-specific primers for ARL4 (forward primer, SEQ ID NO.

19 and reverse primer, SEQ ID NO. 20) are used for PCR. Reaction conditions are: 2 minutes of 94°C, 35 cycles with 30 seconds at 94°C, 30 seconds at 53°C, 90 seconds at 72°C, followed by 7 minutes at 72°C with Taq DNA-polymerase. The PCR product is separated on a 2% agarose gel, a band of about 600bp is cut out and purified with the QIAEX II extraction kit (Qiagen). This product is digested with BamH1 and Hindlll and cloned into pQE-30 (Qiagen) that is digested with BamHl and Hindlll. A clone, designated pQE/ARL4 with identical sequence to the database entry (acc. U73960) is used for further experiments.

4.2 Expression of ARL4 1 I LB broth including 10011g/ml ampicillin is inoculated with 0.5 ml of an overnight culture of E. coli M15 (pREP4) that carries pQE/ARL4. The culture is incubated at 37°C with vigorous shaking until OD6oo of 0.6. Expression is induced by adding 1 mM IPTG and the culture is grown further for 4 hours. Cells are harvested by centrifugation at 4000xg for 20 minutes at 4°C. Pellet is frozen at-20°C.

Cells are thawed on ice and resuspended in 2 ml/g cell pellet of lysis buffer (50 mM NaH2PO4, pH 8.0,300 mM NaCl, 10 mM imidazole). Then, lysozyme is added to 1 mg/ml and incubated on ice for 30 minutes. Then, cells are sonicated (six bursts of 10 seconds at 300 W). 10 ug/ml RNase A and 5 jug/m ! DNase ! is added and incubated on ice for 10 minutes. Then, lysates are cleared by spinning debris at 10000xg for 20 minutes at 4°C. Then, protease inhibitors (40 pg/ml bacitracin, 4 ßlg/ml leupeptin, 4 Ag/ml chymostatin, 10 ug/ml pefabloc, 100 µM PMSF) are added.

3 ml of Ni-NTA resin (Qiagen) are added to the lysate and filled into a column.

Binding to the resin is allowed for 60 minutes at 4°C during gentle shaking. Then,

column outlet is opened, the resin washed twice with 12 mi wash buffer (50 mM NaH2PO4, pH 8.0,300 mM NaCI, 20 mM imidazole) and eluted with four times 3 ml of elution buffer (50 mM NaH2PO4, pH 8.0,300 mM NaCI, 250 mM imidazole). The elution fraction that contains the recombinant protein is determined by SDS-PAGE and protein concentration of the purified protein is determined by the method of Bradford.

4.3 GTPyS binding assay Recombinant ARL4 (1 pM) is incubated at 37 °C with [35S] GTPS or [3H] GDP (10 uM, -1000 cpm/pmol) in 50 mM Hepes (pH7.5), 1 mM dithiothreitol, 1 mM MgCl2 with or without (as indicated in the figure legends) 2 mM EDTA (1 uM or 1 mM free Mg2+), 100 mM KCI. Substances according to the invention are preincubated with ARL4 for 5 minutes at 4°C in a concentration range from 0.5 to 300 nM before starting the GTPyS binding reaction. At various time points (10 seconds to 30 minutes) samples of 25 pl (25 pmoles of ARF) are removed, diluted into 2 ml of ice-cold 20 mM Hepes (pH 7.5), 100 mM NaCI, and 10 mM MgCl2, and filtered on 25-mm BA 85 nitrocellulose filters (Schleicher & Schull). Filters are washed twice with 2 ml of the same buffer, dried, and quantified by scintillation counting.

Example 5: GNS, A gene identified as being downregulated in COPD smokers compared to healthy smokers is Glucosamine-6-sulphatase (GNS). GNS hydrolysis the 6-sulfate group of the N-acetyl-d-glucosamine 6-sulfate units of heparan (Kresse et al. 1980). GNS is consistently found downregulated (44%) in comparisons between COPD smokers and healthy smokers. This is shown by,, fold change"values (Tab. 4). The p values for two separate groups comparing COPD smokers and healthy smokers are 0.05 and 0.006. Tab. 4: Fold change values (FC) for comparisons between obstructed smoker and healthy smokers. On average is downregulated by-2.0fold, the median is-1.8fold comp FC comp FC comp FC comp FC 1 vs 2 1. 0 5 vs 43-4. 6 39 vs 57-2. 4 68 vs 66-3. 6 1 vs 37 1. 0 5 vs 56-1. 7 39 vs 58-3.3 68 vs 69-2.3 1 vs 43-3. 7 5 vs 57-3. 1 39 vs 62-1. 1 68 vs 76-2. 6 1 vs 56-1. 1 5 vs 58-4. 0 44 vs 2-1. 2 68 vs 78-2.6 1 vs 57-2. 3 5 vs 62 1. 0 44 vs 37-1. 2 70 vs 65-1. 4 1 vs 58-3. 0 6 vs 2 1. 0 44 vs 43-4. 3 70 vs 66-1. 6 1 vs 62 1.0 6 vs 37 1. 1 44 vs 56-1. 3 70 vs 69 1.0 3 vs 2-1. 5 6 vs 43-3. 5 44 vs 57-2. 6 70 vs 76-1. 1 3 vs 37-1. 4 6 vs 56 1. 0 44 vs 58-3. 7 70 vs 78-1. 1 3 vs 43-5. 0 6 vs 57-2. 2 44 vs 62-1. 2 71 vs 65-2. 1 3 vs 56-1. 8 6 vs 58-3. 0 64 vs 65-2. 3 71 vs 66-2.5 3 vs 57-3. 1 6 vs 62 1. 1 64 vs 66-2. 6 71 vs 69-1. 7 3 vs 58-3. 9 39 vs 2 1. 0 64 vs 69-1. 7 71 vs 76-1. 8 3 vs 62-1. 3 39 vs 37-1. 1 64 vs 76-1. 9 71 vs 78-1. 8 5 vs 2-1. 7 39 vs 43-3. 8 64 vs 78-1. 9 5 vs 37-1. 7 39 vs 56 1. 0 68 vs 65-3. 1

The protein is cloned and assays are designed and performed in an analogous manner to the cloning and assays described hereinbefore.

Example 6: Transglutaminase 2 A gene identified as being downregulated in COPD smokers compared to healthy smokers is transglutaminase 2. This enzyme belongs to a family of calcium- dependent transglutaminases that catalyze the covalent cross-linking of specific proteins by the formulation of (y-glutamyl) lysine bonds and the conjugation of polyamines to proteins (Folk 1980). Transglutaminases can also be secreted. The physiological functions are not well understood, it may be involved in the specialized processing of the matrix that occurs during bone formation, wound healing, and other remodeling processes (Lu et al. 1995).

Transglutaminase 2 is consistently found downregulated (55%) in comparisons between COPD smokers and healthy smokers. This is shown by,, fold change" values (Tab. 5). The p values for two separate groups comparing COPD smokers and healthy smokers are 0.04 and 0.16.

Tab. 5: Fold change values (FC) for comparisons between obstructed smoker and healthy smokers. On average is downregulated by 2.3fold, the median is-2.35fold comp FC comp FC comp FC comp FC 1 vs 2 1. 0 5 vs 43-5. 6 39 vs 57-2.3 68 vs 66-2.8 1 vs 37-3. 6 5 vs 56-1. 4 39 vs 58-3.9 68 vs 69-7.4 1 vs 43-6. 9 5 vs 57-3. 7 39 vs 62 1. 0 68 vs 76-4-4 1 vs 56-1. 5 5 vs 58-7. 5 44 vs 2 1. 0 68 vs 78-3. 4 1 vs 57-3. 6 5 vs 62 1.0 44 vs 37-3.2 70 vs 65 1. 5 1 vs 58-8. 9 6 vs 2 2. 2 44 vs 43-7. 7 70 vs 66 1.2 1 vs 62 1. 0 6 vs 37-2. 2 44 vs 56-1. 9 70 vs 69-2. 5 3 vs 2 1. 0 6 vs 43-3. 6 44 vs 57-3. 8 70 vs 76-1. 4 3 vs 37-2. 5 6 vs 56 1. 0 44 vs 58-11. 3 70 vs 78 1. 0 3 vs 43-4. 5 6 vs 57-2. 5 44 vs 62 1.0 71 vs 65-1.8 3 vs 56-1. 2 6 vs 58-4. 7 64 vs 65 1. 4 71 vs 66-2. 4 3 vs 57-2. 8 6 vs 62-1. 2 64 vs 66 1. 1 71 vs 69-6. 9 3 vs 58-5. 9 39 vs 2 1. 0 64 vs 69-2. 7 71 vs 76-3. 9 3 vs 62 1. 0 39 vs 37-1. 8 64 vs 76-1.5 71 vs 78-2. 8 5vs2 1. 0 39vs43-2. 9 64vs78-1. 1 5 vs 37-3. 3 39 vs 56 1. 2 68 vs 65-2. 1 The protein is cloned and assays are designed and performed in an analogous manner to the cloning and assays described hereinbefore.

Example 7: Stearyl-CoA-Desaturase A gene identified as being downregulated in COPD smokers compared to healthy smokers is Stearoyl-CoA-Desaturase. Stearoyl-CoA-Desaturase catalyzes the

oxidation of paimitoyl-CoA and stearoyl-CoA at the A9 position to form the mono- unsaturated fatty acyl-CoA esters, palmitoleoyl-CoA and aoleoyl-CoA, respectively (Enoch et al. 1976).

Stearoyl-CoA-desaturase is consistently found downregulated (48%) in comparisons between COPD smokers and healthy smokers. This is shown by,, fold change" values (Tab. 6). The p values for two separate groups comparing COPD smokers and healthy smokers are 0.03 and 0.15.

Tab. 6: Fold change values (FC) for comparisons between obstructed smoker and healthy smokers. On average is downregulated by 2.3fold, the median is-1.9fold comp FC comp FC comp FC comp FC 1 vs 2-1. 7 5 vs 43-5. 8 39 vs 57-3.9 68 vs 66-2.5 1 vs 37 1.0 5 vs 56-2. 1 39 vs 58-7. 3 68 vs 69-1.2 1 vs 43-4. 0 5 vs 57-3. 7 39 vs 62-1. 8 68 vs 76-1. 2 1 vs 56 1. 0 5 vs 58-6. 5 44 vs 2-1. 1 68 vs 78-1. 5 1 vs 57-2. 4 5 vs 62-2. 3 44 vs 37 1. 3 70 vs 65-1. 5 1 vs 58-4. 6 6 vs 2-3. 0 44 vs 43-2. 4 70 vs 66-1. 2 1 vs 62-1. 1 6 vs 37-1. 8 44 vs 56 1.4 70 vs 69 1.5 3 vs 2-1. 8 6 vs 43-7. 1 44 vs 57-1.5 70 vs 76 1. 5 3 vs 37-1. 1 6 vs 56-2. 2 44 vs 58-2. 9 70 vs 78 1.3 3 vs 43-4. 4 6 vs 57-4. 3 44 vs 62 1.3 71 vs 65-2.5 3 vs 56-1. 2 6 vs 58-8. 2 64 vs 65-4. 2 71 vs 66-1. 9 3 vs 57-2. 7 6 vs 62-2. 4 64 vs 66-3.3 71 vs 69 1.0 3 vs 58-5. 0 39 vs 2-2. 7 64 vs 69-1.7 71 vs 76-1.1 3 vs 62-1. 2 39 vs 37-1. 6 64 vs 76-1. 7 71 vs 78-1. 3 5 vs 2-2. 9 39 vs 43-6. 4 64 vs 78-2. 2 5 vs 37-1. 9 39 vs 56-1. 7 68 vs 65-3.3

The protein is cloned and assays are designed and performed in an analogous manner to the cloning and assays described hereinbefore.

Example 8 : UDP-Glucose Ceramide Glycosyltransferase A gene identified as being downregulated in COPD smokers compared to healthy smokers is UDP-glucose Ceramide Glucosyltransferase. This enzyme catalyzes the transfer of glucose from UDP-glucose to ceramide. The product glucosyl-ceramid serces as the core structure of more than 300 glycoshingolipids that are involved in multiple cellular processes as differentiation, adhesion, proliferation, and cell-cell recognition (Basu et al. 1968, Ichikawa et al. 1996).

Ceramide Glucosyltransferase is consistently found downregulated (48%) in comparisons between COPD smokers and healthy smokers. This is shown by,, fold change"values (Tab. 7).

Tab. 7: Fold change values (FC) for comparisons between obstructed smoker and healthy smokers. On average is downregulated by 1.2fold, the median is-1.9fold comp FC comp FC comp FC comp FC 1 vs 2 1.3 5 vs 43 -2.4 39 vs 57 -1.6 68 vs 66-4.0 1 vs 37-2. 4 5 vs 56-2. 0 39 vs 58-2.6 68 vs 69-1.1 1 vs 43-1. 9 5 vs 57-1. 6 39 vs 62-2. 3 68 vs 76-2.9 1 vs 56-1. 5 5 vs 58-2. 6 44 vs 2 7. 2 68 vs 78-3.4 1 vs57-1. 3 5vs62-2. 0 44vs37 1. 9 70 vs 65 1.0 1 vs 58-2. 1 6 vs 2 1. 0 44 vs 43 2.7 70 vs 66-2. 0 1 vs 62-1. 5 6 vs 37-4. 2 44 vs 56 3. 5 70 vs 69 1. 5 3 vs 2 1. 3 6 vs 43-2. 8 44 vs 57 4. 6 70 vs 76-1. 4 3 vs 37 -2.6 6 vs 56 -2.3 44 vs 58 2.7 70 vs 78 -1. 8 3 vs 43-1. 9 6 vs 57-1. 8 44 vs 62 3.4 71 vs 65-2. 0 3 vs 56-1. 6 6 vs 58-3. 0 64 vs 65-1. 7 71 vs 66-4. 3 3 vs 57-1. 3 6 vs 62-2. 4 64 vs 66-3. 2 71 vs 69 1.0 3 vs 58-2. 1 39 vs 2 1.0 64 vs 69-1. 1 71 vs 76-2. 5 3 vs 62-1. 7 39 vs 37-3. 5 64 vs 76-2. 5 71 vs 78-3. 7 5 vs 2 1.0 39 vs 43 -2.4 64 vs 78 -2. 9 5 vs 37-3. 1 39 vs 56-2. 2 68 vs 65-1. 9

The protein is cloned and assays are designed and performed in an analogous manner to the cloning and assays described hereinbefore.

Literature: MIF Calandra, T., Bernhagen, J., Mitchell, R. A., and Bucala, R. (1994). J. Exp. Med. 179, 1985-1902.

Bernhagen, J., Calandra, T., and Bucala, R. (1998). J. Mol. Med. 76,151-161.

Calandra, T., Echtenacher, B., Le Roy, D. Pugin, J., Metz, C. N., Hultner, L., Heumann, D., Männel, D., Bucala, R., and Glauser, M. P. (2000). Nat. Med. 6,164- 170.

DAD 1 Nakashima, T., Sekiguchi, T., Kuraoka, A., Fukushima, K., Shibata, Y., Komiyama, S., Nishimoto, T. (1993). Mol. Cell. Biol. 13,6367-6374.

Kelleher, D., and Gilmore, R. (1997). Proc. Natl. Acad. Sci. U. S. A. 94, 4994-4999.

ARL4 Jacobs, S., Schilf, C., Fliegert, F., Koling, S., Weber, Y., Schürmann, A., and Joost, H.-G. (1999). FEBS Lett. 456,384-388.

GNS Kresse, H., Paschke, E., von Figura, K., Gilberg, W., and Fuchs, W. (1980). Proc.

Natl. Acad. Sci. U. S. A. 77,6822-6826.

Transglutaminase 2 Folk, J. E. (1980). Annu. Rev. Biochem. 49,517-531 Lu, S., Saydak, M., Gentile, V., Stein, J. P., and Davies, P. J. A. (1995). J. Biol. Chem.

270,9748-9756.

Stearoyl-CoA-Desaturase Enoch, H. G., Catala, A., and Strittmater, P. (1976). J. Biol. Chem. 251,5095-5103.

UDP-glucose Ceramide Glucosyltransferase Basu, S., Kaufmann, B., and Rosemann, S. (1968). J. Biol. Chem. 243,5802-5807.

Ichikawa, S., Sakiyama, H., Suzuki, G., Jwa Hidari, K. I.-P., and Hirabayashi, Y.

(1996). Proc. Natl. Acad. Sci. U. S. A. 93,4638-4643.

Cell lines Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., and Tada, K.

(1980). Int. J. Cancer 26,171-176.

Ziegler-Heitbrock, H. W., Thiel, E., Futterer, A., Herzog, V., Wirtz, A., and Riethmüller, G. (1988). Int. J. Cancer 41, 456-461.

SEQUENCE LISTING <110> Boehringer Ingelheim Pharma KG <120> Method for identifying substances which positively influence inflammatory conditions of chronic inflammatory airway diseases <130> COPDrestlicheP <140> <141> <150> US 60/257,878 <151> 2000-12-22 <160> 20 <170> PatentIn Ver. 2.1 <210> 1 <211> 2167 <212> DNA <213> Homo sapiens <400> 1 ctgcaggaac caatacccat aggctatttg tataaatggg ccatggggcc tcccagctgg 60 aggctggctg gtgccacgag ggtcccacag gcatgggtgt ccttcctata tcacatggcc 120 ttcactgaga ctggtatatg gattgcacct atcagagacc aaggacagga cctccctgga 180 aatctctgag gacctggcct gtgatccagt tgctgccttg tcctcttcct gctatgtcat 240 ggcttatctt ctttcaccca ttcattcatt cattcattca ttcagcagta ttagtcaatg 300 tctcttgata tgcctggcac ctgctagatg gtccccgagt ttaccattag tggaaaagac 360 atttaagaaa ttcaccaagg gctctatgag aggccataca cggtggacct gactagggtg 420 tggcttccct gaggagctga agttgcccag aggcccagag aaggggagct gagcacgttt 480 gaaccactga acctgctctg gacctcgcct ccttccttcg gtgcctccca gcatcctatc 540 ctctttaaag agcaggggtt cagggaagtt ccctggatgg tgattcgcag gggcagctcc 600 cctctcacct gccgcatgac taccccgccc catctcaaac acacaagctc acgcatgcgg 660 gactggagcc cttgaggaca tgtggcccaa agacaggagg tacaggggct cagtgcgtgc 720 agtggaatga actgggcttc atctctggaa gggtaagggg ccatcttccg ggttcaccgc 780

cgcatcccca cccccggcac agcgcctcct ggcgactaac atcggtgact tagtgaaagg 840 actaagaaag acccgaggcg aggccggaac aggccgattt ctagccgcca agtggagaac 900 aggttggagc ggtgcgccgg gcttagcggc ggttgctgga ggaacgggcg gagtcgccca 960 gggtcctgcc ctgcgggggt cgagccgagg caggcggtga cttccccact cggggcggag 1020 ccgcagcctc gcgggggcgg ggcctggcgc cggcggtggc gtcacaaaag gcgggaccac 1080 agtggtgtcc gagaagtcag gcacgtagct cagcggcggc cgcggcgcgt gcgtctgtgc 1140 ctctgcgcgg gtctcctggt ccttctgcca tcatgccgat gttcatcgta aacaccaacg 1200 tgccccgcgc ctccgtgccg gacgggttcc tctccgagct cacccagcag ctggcgcagg 1260 ccaccggcaa gcccccccag gtttgccggg aggggacagg aagagggggg tgcccaccgg 1320 acgaggggtt ccgcgctggg agctggggag gcgactcctg aacggagctg gggggcgggg 1380 cggggggagg acggtggctc gggcccgaag tggacgttcg gggcccgacg aggtcgctgg 1440 ggcgggctga ccgcgccctt tcctcgcagt acatcgcggt gcacgtggtc ccggaccagc 1500 tcatggcctt cggcggctcc agcgagccgt gcgcgctctg cagcctgcac agcatcggca 1560 agatcggcgg cgcgcagaac cgctcctaca gcaagctgct gtgcggcctg ctggccgagc 1620 gcctgcgcat cagcccggac aggtacgcgg agtcgcggag gggcggggga ggggcggcgg 1680 cgcgcggcca ggcccgggac tgagccaccc gctgagtccg gcctcctccc cccgcagggt 1740 ctacatcaac tattacgaca tgaacgcggc caatgtgggc tggaacaact ccaccttcgc 1800 ctaagagccg cagggaccca cgctgtctgc gctggctcca cccgggaacc cgccgcacgc 1860 tgtgttctag gcccgcccac cccaaccttc tggtggggag aaataaacgg tttagagact 1920 aggagtgcct cggggttcct tggcttgcgg gaggaattgg tgcagagccg ggacattggg 1980 gagcgaggtc gggaaacggt gttgggggcg ggggtcaggg ccgggttgct ctcctcgaac 2040 ctgctgttcg ggagcccttt tgtccagcct gtccctccta cgctcctaac agaggagccc 2100 cagtgtcttt ccattctatg gcgtacgaag ggatgaggag aagttggcac tctgccctgg 2160 gctgcag 2167 <210> 2 <211> 115 <212> PRT <213> Homo sapiens <400> 2 Met Pro Met Phe Ile Val Asn Thr Asn Val Pro Arg Ala Ser Val Pro 1 5 10 15 Asp Gly Phe Leu Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly 20 25 30 Lys Pro Pro Gln Tyr Ile Ala Val His Val Val Pro Asp Gln Leu Met 35 40 45

Ala Phe Gly Gly Ser Ser Glu Pro Cys Ala Leu Cys Ser Leu His Ser 50 55 60 Ile Gly Lys Ile Gly Gly Ala Gln Asn Arg Ser Tyr Ser Lys Leu Leu 65 70 75 80 Cys Gly Leu Leu Ala Glu Arg Leu Arg Ile Ser Pro Asp Arg Val Tyr 85 90 95 Ile Asn Tyr Tyr Asp Met Asn Ala Ala Asn Val Gly Trp Asn Asn Ser 100 105 110 Thr Phe Ala 115 <210> 3 <211> 699 <212> DNA <213> Homo sapiens <400> 3 catccggtgt ggtcgacggg tcctccaaga gtttggggcg cggaccggag taccttgcgt 60 gcagttatgt cggcgtcggt agtgtctgtc atttcgcggt tcttagaaga gtacttgagc 120 tccactccgc agcgtctgaa gttgctggac gcgtacctgc tgtatatact gctgaccggg 180 gcgctgcagt tcggttactg tctcctcgtg gggaccttcc ccttcaactc ttttctctcg 240 ggcttcatct cttgtgtggg gagtttcatc ctagcggttt gcctgagaat acagatcaac 300 ccacagaaca aagcggattt ccaaggcatc tccccagagc gagcctttgc tgattttctc 360 tttgccagca ccatcctgca ccttgttgtc atgaactttg ttggctgaat cattctcatt 420 tacttaattg aggagtagga gactaaaaga atgttcactc tttgaatttc ctggataaga 480 gttctggaga tggcagctta ttggacacat ggattttctt cagatttgac acttactgct 540 agctctgctt tttatgacag gagaaaagcc cagagttcac tgtgtgtcag aacaactttc 600 taacaaacat ttattaatcc agcctctgcc tttcattaaa tgtaaccttt tgctttccaa 660 attaaagaac tccatgccac tcctcaaaaa aaaaaaaaa 699 <210> 4 <211> 113 <212> PRT

<213> Homo sapiens <400> 4 Met Ser Ala Ser Val Leu Ser Val Ile Ser Arg Phe Leu Glu Glu Tyr 1 5 10 15 Leu Ser Ser Thr Pro Gln Arg Leu Lys Leu Leu Asp Ala Tyr Leu Leu 20 25 30 Tyr Ile Leu Leu Thr Gly Ala Leu Gln Phe Gly Tyr Cys Leu Leu Val 35 40 45 Gly Thr Phe Pro Phe Asn Ser Phe Leu Ser Gly Phe Ile Ser Cys Val 50 55 60 Gly Ser Phe Ile Leu Ala Val Cys Leu Arg Ile Gln Ile Asn Pro Gln 65 70 75 80 Asn Lys Ala Asp Phe Gln Gly Ile Ser Pro Glu Arg Ala Phe Ala Asp 85 90 95 Phe Leu Phe Ala Ser Thr Ile Leu His Leu Val Val Met Asn Phe Val 100 105 110 Gly <210> 5 <211> 1077 <212> DNA <213> Homo sapiens <400> 5 cttatccctg cgtagaaacg cctgccaatg ctttctcatt tggacccaga ctccagatcg 60 ggagcagtct tatagctgga tcagctacca agagaagttg taaaccaaga agagaaaagc 120 atttcaattt gggacattta tttgcacctg gaaatgggga atgggctgtc agaccagact 180

tctatcctgt ccaacctgcc ttcatttcag tctttccaca ttgttattct gggtttggac 240 tgtgctggaa agacaacagt cttatacagg ctgcagttca atgaatttgt aaataccgta 300 cctaccaaag gatttaacac tgagaaaatt aaggtaacct tgggaaattc taaaacagtc 360 acttttcact tctgggatgt aggtggtcag gagaaattaa ggccactgtg gaagtcatat 420 accagatgca cagatggcat tgtatttgtt gtggactctg ttgatgtcga aaggatggaa 480 gaagccaaaa ctgaacttca caaaataact aggatatcag aaaatcaggg agtccctgta 540 cttatagttg ctaacaaaca agatttgagg aactcattgt cactttcaga aattgagaaa 600 ttgttagcaa tgggtgaact gagctcatca actccttggc atttgcagcc tacctgtgca 660 atcataggag atggcctaaa ggaaggactt gagaaactac atgatatgat cattaaaaga 720 agaaaaatgt tgcggcaaca gaaaaagaaa agatgaatat caatacctat tatatctgtg 780 tggagtaggt tttctctggt ctgattttga caaatagaag agtgtctaca ccgtcctttg 840 cctgtctgcc ctcctggatg ctattaaagc tttgttttgt tgaacaatca gatgcccaac 900 tctgttgcct tgtggaagat gagtaaatgc agtgcttctt aaagtggtct cttctcccta 960 ccccacaaat cttttggtac taccatttgg ggaagccaag caaggatagt aaattgacca 1020 gaacacagtt gtgggaattt ggtctgaagt tagtgaaata aaactttaaa gagtgtc 1077 <210> 6 <211> 200 <212> PRT <213> Homo sapiens <400> 6 Met Gly Asn Gly Leu Ser Asp Gln Thr Ser Ile Leu Ser Asn Leu Pro 1 5 10 15 Ser Phe Gln Ser Phe His Ile Val Ile Leu Gly Leu Asp Cys Ala Gly 20 25 30 Lys Thr Thr Val Leu Tyr Arg Leu G1n Phe Asn Glu Phe Val Asn Thr 35 40 45 Val Pro Thr Lys Gly Phe Asn Thr Glu Lys Ile Lys Val Thr Leu Gly 50 55 60 Asn Ser Lys Thr Val Thr Phe His Phe Trp Asp Val Gly Gly Gln Glu 65 70 75 80

Lys Leu Arg Pro Leu Trp Lys Ser Tyr Thr Arg Cys Thr Asp Gly Ile 85 90 95 Val Phe Val Val Asp Ser Val Asp Val Glu Arg Met Glu Glu Ala Lys 100 105 110 Thr Glu Leu His Lys Ile Thr Arg Ile Ser Glu Asn Gln Gly Val Pro 115 120 125 Val Leu Ile Val Ala Asn Lys Gln Asp Leu Arg Asn Ser Leu Ser Leu 130 135 140 Ser Glu Ile Glu Lys Leu Leu Ala Met Gly Glu Leu Ser Ser Ser Thr 145 150 155 160 Pro Trp His Leu Gln Pro Thr Cys Ala Ile Ile Gly Asp Gly Leu Lys 165 170 175 Glu Gly Leu Glu Lys Leu His Asp Met Ile Ile Lys Arg Arg Lys Met 180 185 190 Leu Arg Gln Gln Lys Lys Lys Arg 195 200 <210> 7 <211> 2379 <212> DNA <213> Homo sapiens <400> 7 ggaattccgg tcggcctctc gcccttcagc tacctgtgcg tccctccgtc ccgtcccgtc 60 ccggggtcac cccggagcct gtccgctatg cggctcctgc ctctagcccc aggtcggctc 120 cggcggggca gcccccgcca cctgccctcc tgcagcccag cgctgctact gctggtgctg 180 ggcggctgcc tgggggtctt cggggtggct gcgggaaccc ggaggcccaa cgtggtgctg 240 ctcctcacgg acgaccagga cgaagtgctc ggcggcatga caccactaaa gaaaaccaaa 300 gctctcatcg gagagatggg gatgactttt tccagtgctt atgtgccaag tgctctctgc 360 tgccccagca gagccagtat cctgacagga aagtacccac ataatcatca cgttgtgaac 420

aacactctgg aggggaactg cagtagtaag tcctggcaga agatccaaga accaaatact 480 ttcccagcaa ttctcagatc aatgtgtggt tatcagacct tttttgcagg gaaatattta 540 aatgagtacg gagccccaga tgcaggtgga ctagaacacg ttcctctggg ttggagttac 600 tggtatgcct tggaaaagaa ttctaagtat tataattaca ccctgtctat caatgggaag 660 gcacggaagc atggtgaaaa ctatagtgtg gactacctga cagatgtttt ggctaatgtc 720 tccttggact ttctggacta caagtccaac tttgagccct tcttcatgat gatcgccact 780 ccagcgcctc attcgccttg gacagctgca cctcagtacc agaaggcttt ccagaatgtc 840 tttgcaccaa gaaacaagaa cttcaacatc catggaacga acaagcactg gttaattagg 900 caagccaaga ctccaatgac taattcttca atacagtttt tagataatgc atttaggaaa 960 aggtggcaaa ctctcctctc agttgatgac cttgtggaga aactggtcaa gaggctggag 1020 ttcactgggg agctcaacaa cacttacatc ttctatacct cagacaatgg ctatcacaca 1080 ggacagtttt ccttgccaat agacaagaga cagctgtatg agtttgatat caaagttcca 1140 ctgttggttc gaggacctgg gatcaaacca aatcagacaa gcaagatgct ggttgccaac 1200 attgacttgg gtcctactat tttggacatt gctggctacg acctaaataa gacacagatg 1260 gatgggatgt ccttattgcc cattttgaga ggtgccagta acttgacctg gcgatcagat 1320 gtcctggtgg aataccaagg agaaggccgt aacgtcactg acccaacatg cccttccctg 1380 agtcctggcg tatctcaatg cttcccagac tgtgtatgtg aagatgctta taacaatacc 1440 tatgcctgtg tgaggacaat gtcagcattg tggaatttgc agtattgcga gtttgatgac 1500 caggaggtgt ttgtagaagt ctataatctg actgcagacc cagaccagat cactaacatt 1560 gctaaaacca tagacccaga gcttttagga aagatgaact atcggttaat gatgttacag 1620 tcctgttctg ggccaacctg tcgcactcca ggggtttttg accccggata caggtttgac 1680 ccccgtctca tgttcagcaa tcgcggcagt gtcaggactc gaagattttc caaacatctt 1740 ctgtagcgac ctcacacagc ctctgcagat ggatccctgc acgcctcttt ctgatgaagt 1800 gattgtagta ggtgtctgta gctagtcttc aagaccacac ctggaagagt ttctgggctg 1860 gctttaagtc ctgtttgaaa aagcaaccca gtcagctgac ttcctcgtgc aatgtgttaa 1920 actgtgaact ctgcccatgt gtcaggagtg gctgtctctg gtctcttcct ttagctgaca 1980 aggacactcc tgaggtcttt gttctcactg tatttttttt atcctggggc cacagttctt 2040 gattattcct cttgtggtta aagactgaat ttgtaaaccc attcagataa atggcagtac 2100 tttaggacac acacaaacac acagatacac cttttgatat gtaagcttga cctaaagtca 2160 aaggacctgt gtagcatttc agattgagca cttcactatc aaaaatacta acatcacatg 2220 gcttgaagag taaccatcag agctgaatca tccaagtaag aacaagtacc attgttgatt 2280 gataagtaga gatacatttt ttatgatgtt catcacagtg tggtaaggtt gcaaattcaa 2340 aacatgtcac ccaagctctg ttcatgtttt tgtgaattc 2379 <210> 8 <211> 552 <212> PRT <213> Homo sapiens <400> 8

Met Arg Leu Leu Pro Leu Ala Pro Gly Arg Leu Arg Arg Gly Ser Pro 1 5 10 15 Arg His Leu Pro Ser Cys Ser Pro Ala Leu Leu Leu Leu Val Leu Gly 20 25 30 Gly Cys Leu Gly Val Phe Gly Val Ala Ala Gly Thr Arg Arg Pro Asn 35 40 45 Val Val Leu Leu Leu Thr Asp Asp Gln Asp Glu Val Leu Gly Gly Met 50 55 60 Thr Pro Leu Lys Lys Thr Lys Ala Leu Ile Gly Glu Met Gly Met Thr 65 70 75 80 Phe Ser Ser Ala Tyr Val Pro Ser Ala Leu Cys Cys Pro Ser Arg Ala 85 90 95 Ser Ile Leu Thr Gly Lys Tyr Pro His Asn His His Val Val Asn Asn 100 105 110 Thr Leu Glu Gly Asn Cys Ser Ser Lys Ser Trp Gln Lys Ile Gln Glu 115 120 125 Pro Asn Thr Phe Pro Ala Ile Leu Arg Ser Met Cys Gly Tyr Gln Thr 130 135 140 Phe Phe Ala Gly Lys Tyr Leu Asn Glu Tyr Gly Ala Pro Asp Ala Gly 145 150 155 160 Gly Leu Glu His Val Pro Leu Gly Trp Ser Tyr Trp Tyr Ala Leu Glu 165 170 175 Lys Asn Ser Lys Tyr Tyr Asn Tyr Thr Leu Ser Ile Asn Gly Lys Ala 180 185 190

Arg Lys His Gly Glu Asn Tyr Ser Val Asp Tyr Leu Thr Asp Val Leu 195 200 205 Ala Asn Val Ser Leu Asp Phe Leu Asp Tyr Lys Ser Asn Phe Glu Pro 210 215 220 Phe Phe Met Met Ile Ala Thr Pro Ala Pro His Ser Pro Trp Thr Ala 225 230 235 240 Ala Pro Gln Tyr Gln Lys Ala Phe Gln Asn Val Phe Ala Pro Arg Asn 245 250 255 Lys Asn Phe Asn Ile His Gly Thr Asn Lys His Trp Leu Ile Arg Gln 260 265 270 Ala Lys Thr Pro Met Thr Asn Ser Ser Ile Gln Phe Leu Asp Asn Ala 275 280 285 Phe Arg Lys Arg Trp Gln Thr Leu Leu Ser Val Asp Asp Leu Val Glu 290 295 300 Lys Leu Val Lys Arg Leu Glu Phe Thr Gly Glu Leu Asn Asn Thr Tyr 305 310 315 320 Ile Phe Tyr Thr Ser Asp Asn Gly Tyr His Thr Gly Gln Phe Ser Leu 325 330 335 Pro Ile Asp Lys Arg Gln Leu Tyr Glu Phe Asp Ile Lys Val Pro Leu 340 345 350 Leu Val Arg Gly Pro Gly Ile Lys Pro Asn Gln Thr Ser Lys Met Leu 355 360 365 Val Ala Asn Ile Asp Leu Gly Pro Thr Ile Leu Asp Ile Ala Gly Tyr 370 375 380

Asp Leu Asn Lys Thr Gln Met Asp Gly Met Ser Leu Leu Pro Ile Leu 385 390 395 400 Arg Gly Ala Ser Asn Leu Thr Trp Arg Ser Asp Val Leu Val Glu Tyr 405 410 415 Gln Gly Glu Gly Arg Asn Val Thr Asp Pro Thr Cys Pro Ser Leu Ser 420 425 430 Pro Gly Val Ser Gln Cys Phe Pro Asp Cys Val Cys Glu Asp Ala Tyr 435 440 445 Asn Asn Thr Tyr Ala Cys Val Arg Thr Met Ser Ala Leu Trp Asn Leu 450 455 460 Gln Tyr Cys Glu Phe Asp Asp Gln Glu Val Phe Val Glu Val Tyr Asn 465 470 475 480 Leu Thr Ala Asp Pro Asp Gln Ile Thr Asn Ile Ala Lys Thr Ile Asp 485 490 495 Pro Glu Leu Leu Gly Lys Met Asn Tyr Arg Leu Met Met Leu Gln Ser 500 505 510 Cys Ser Gly Pro Thr Cys Arg Thr Pro Gly Val Phe Asp Pro Gly Tyr 515 520 525 Arg Phe Asp Pro Arg Leu Met Phe Ser Asn Arg Gly Ser Val Arg Thr 530 535 540 Arg Arg Phe Ser Lys His Leu Leu 545 550 <210> 9 <211> 3257

<212> DNA <213> Homo sapiens <400> 9 aacaggcgtg acgccagttc taaacttgaa acaaaacaaa acttcaaagt acaccaaaat 60 agaacctcct taaagcataa atctcacgga gggtctcggc cgccagtgga aggagccacc 120 gcccccgccc cgaccatggc cgaggagctg gtcttagaga ggtgtgatct ggagctggag 180 accaatggcc gagaccacca cacggccgac ctgtgccggg agaagctggt ggtgcgacgg 240 ggccagccct tctggctgac cctgcacttt gagggccgca actaccaggc cagtgtagac 300 agtctcacct tcagtgtcgt gaccggccca gcccctagcc aggaggccgg gaccaaggcc 360 cgttttccac taagagatgc tgtggaggag ggtgactgga cagccaccgt ggtggaccag 420 caagactgca ccctctcgct gcagctcacc accccggcca acgcccccat cggcctgtat 480 cgcctcagcc tggaggcctc cactggctac cagggatcca gctttgtgct gggccacttc 540 attttgctct tcaacgcctg gtgcccagcg gatgctgtgt acctggactc ggaagaggag 600 cggcaggagt atgtcctcac ccagcagggc tttatctacc agggctcggc caagttcatc 660 aagaacatac cttggaattt tgggcagttt caagatggga tcctagacat ctgcctgatc 720 cttctagatg tcaaccccaa gttcctgaag aacgccggcc gtgactgctc ccggcgcagc 780 agccccgtct acgtgggccg ggtgggtagt ggcatggtca actgcaacga tgaccagggt 840 gtgctgctgg gacgctggga caacaactac ggggacggcg tcagccccat gtcctggatc 900 ggcagcgtgg acatcctgcg gcgctggaag aaccacggct gccagcgcgt caagtatggc 960 cagtgctggg tcttcgccgc cgtggcctgc acagtgctga ggtgcctagg catccctacc 1020 cgcgtcgtga ccaactacaa ctcggcccat gaccagaaca gcaaccttct catcgagtac 1080 ttccgcaatg agtttgggga gatccagggt gacaagagcg agatgatctg gaacttccac 1140 tgctgggtgg agtcgtggat gaccaggccg gacctgcagc cggggtacga gggctggcag 1200 gccctggacc caacgcccca ggagaagagc gaaggaacgt actgctgtgg cccagttcca 1260 gttcgtgcca tcaaggaggg cgacctgagc accaagtacg atgcgccctt tgtctttgcg 1320 gaggtcaatg ccgacgtggt agactggatc cagcaggacg atgggtctgt gcacaaatcc 1380 atcaaccgtt ccctgatcgt tgggctgaag atcagcacta agagcgtggg ccgagacgag 1440 cgggaggata tcacccacac ctacaaatac ccagaggggt cctcagagga gagggaggcc 1500 ttcacaaggg cgaaccacct gaacaaactg gccgagaagg aggagacagg gatggccatg 1560 cggatccgtg tgggccagag catgaacatg ggcagtgact ttgacgtctt tgcccacatc 1620 accaacaaca ccgctgagga gtacgtctgc cgcctcctgc tctgtgcccg caccgtcagc 1680 tacaatggga tcttggggcc cgagtgtggc accaagtacc tgctcaacct aaccctggag 1740 cctttctctg agaagagcgt tcctctttgc atcctctatg agaaataccg tgactgcctt 1800 acggagtcca acctcatcaa ggtgcgggcc ctcctcgtgg agccagttat caacagctac 1860 ctgctggctg agagggacct ctacctggag aatccagaaa tcaagatccg gatccttggg 1920 gagcccaagc agaaacgcaa gctggtggct gaggtgtccc tgcagaaccc gctccctgtg 1980 gccctggaag gctgcacctt cactgtggag ggggccggcc tgactgagga gcagaagacg 2040 gtggagatcc cagaccccgt ggaggcaggg gaggaagtta aggtgagaat ggacctcgtg 2100 ccgctccaca tgggcctcca caagctggtg gtgaacttcg agagcgacaa gctgaaggct 2160 gtgaagggct tccggaatgt catcattggc cccgcctaag ggacccctgc tcccagcctg 2220 ctgagagccc ccaccttgat cccaatcctt atcccaagct agtgagcaaa atatgcccct 2280 tattgggccc cagaccccag ggcagggtgg gcagcctatg ggggctctcg gaaatggaat 2340 gtgcccctgg cccatctcag cctcctgagc ctgtgggtcc ccactcaccc cctttgctgt 2400

gaggaatgct ctgtgccaga aacagtggga gccctgacct gtgctgactg gggctggggt 2460 gagagaggaa agacctacat tccctctcct gcccagatgc cctttggaaa gccattgacc 2520 acccaccata ttgtttgatc tacttcatag ctccttggag caggcaaaaa agggacagca 2580 tgcccttggc tggatcagga atccagctcc ctagactgca tcccgtacct cttcccatga 2640 ctgcacccag ctccaggggc ccttgggaca cccagagctg ggtggggaca gtgataggcc 2700 caaggtcccc tccacatccc agcagcccaa gcttaatagc cctccccctc aacctcacca 2760 ttgtgaagca cctactatgt gctgggtgcc tcccacactt gctggggctc acggggcctc 2820 caacccattt aatcaccatg ggaaactgtt gtgggcgctg cttccaggat aaggagactg 2880 aggcttagag agaggaggca gccccctcca caccagtggc ctcgtggtta taagcaaggc 2940 tgggtaatgt gaaggcccaa gagcagagtc tgggcctctg actctgagtc cactgctcca 3000 tttataaccc cagcctgacc tgagactgtc gcagaggctg tctggggcct ttatcaaaaa 3060 aagactcagc caagacaagg aggtagagag gggactgggg gactgggagt cagagccctg 3120 gctgggttca ggtcccacgt ctggccagcg actgccttct cctctctggg cctttgtttc 3180 cttgttggtc agaggagtga ttgaacctgc tcatctccaa ggatcctctc cactccatgt 3240 ttgcaataca caattcc 3257 <210> 10 <211> 687 <212> PRT <213> Homo sapiens <400> 10 Met Ala Glu Glu Leu Val Leu Glu Arg Cys Asp Leu Glu Leu Glu Thr 1 5 10 15 Asn Gly Arg Asp His His Thr Ala Asp Leu Cys Arg Glu Lys Leu Val 20 25 30 Val Arg Arg Gly Gln Pro Phe Trp Leu Thr Leu His Phe Glu Gly Arg 35 40 45 Asn Tyr Gln Ala Ser Val Asp Ser Leu Thr Phe Ser Val Val Thr Gly 50 55 60 Pro Ala Pro Ser Gln Glu Ala Gly Thr Lys Ala Arg Phe Pro Leu Arg 65 70 75 80

Asp Ala Val Glu Glu Gly Asp Trp Thr Ala Thr Val Val Asp Gln Gln 85 90 95 Asp Cys Thr Leu Ser Leu Gln Leu Thr Thr Pro Ala Asn Ala Pro Ile 100 105 110 Gly Leu Tyr Arg Leu Ser Leu Glu Ala Ser Thr Gly Tyr G1n Gly Ser 115 120 125 Ser Phe Val Leu Gly His Phe Ile Leu Leu Phe Asn Ala Trp Cys Pro 130 135 140 Ala Asp Ala Val Tyr Leu Asp Ser Glu Glu Glu Arg Gln Glu Tyr Val 145 150 155 160 Leu Thr Gln Gln Gly Phe Ile Tyr Gln Gly Ser Ala Lys Phe Ile Lys 165 170 175 Asn Ile Pro Trp Asn Phe Gly Gln Phe Gln Asp Gly Ile Leu Asp Ile 180 185 190 Cys Leu Ile Leu Leu Asp Val Asn Pro Lys Phe Leu Lys Asn Ala Gly 195 200 205 Arg Asp Cys Ser Arg Arg Ser Ser Pro Val Tyr Val Gly Arg Val Gly 210 215 220 Ser Gly Met Val Asn Cys Asn Asp Asp Gln Gly Val Leu Leu Gly Arg 225 230 235 240 Trp Asp Asn Asn Tyr Gly Asp Gly Val Ser Pro Met Ser Trp Ile Gly 245 250 255 Ser Val Asp Ile Leu Arg Arg Trp Lys Asn His Gly Cys Gln Arg Val 260 265 270

Lys Tyr Gly Gln Cys Trp Val Phe Ala Ala Val Ala Cys Thr Val Leu 275 280 285 Arg Cys Leu Gly Ile Pro Thr Arg Val Val Thr Asn Tyr Asn Ser Ala 290 295 300 His Asp Gln Asn Ser Asn Leu Leu Ile Glu Tyr Phe Arg Asn Glu Phe 305 310 315 320 Gly Glu Ile Gln Gly Asp Lys Ser Glu Met Ile Trp Asn Phe His Cys 325 330 335 Trp Val Glu Ser Trp Met Thr Arg Pro Asp Leu Gln Pro Gly Tyr Glu 340 345 350 Gly Trp Gln Ala Leu Asp Pro Thr Pro Gln Glu Lys Ser Glu Gly Thr 355 360 365 Tyr Cys Cys Gly Pro Val Pro Val Arg Ala Ile Lys Glu Gly Asp Leu 370 375 380 Ser Thr Lys Tyr Asp Ala Pro Phe Val Phe Ala Glu Val Asn Ala Asp 385 390 395 400 Val Val Asp Trp Ile Gln Gln Asp Asp Gly Ser Val His Lys Ser Ile 405 410 415 Asn Arg Ser Leu Ile Val Gly Leu Lys Ile Ser Thr Lys Ser Val Gly 420 425 430 Arg Asp Glu Arg Glu Asp Ile Thr His Thr Tyr Lys Tyr Pro Glu Gly 435 440 445 Ser Ser Glu Glu Arg Glu Ala Phe Thr Arg Ala Asn His Leu Asn Lys 450 455 460

Leu Ala Glu Lys Glu Glu Thr Gly Met Ala Met Arg Ile Arg Val Gly 465 470 475 480 Gln Ser Met Asn Met Gly Ser Asp Phe Asp Val Phe Ala His Ile Thr 485 490 495 Asn Asn Thr Ala Glu Glu Tyr Val Cys Arg Leu Leu Leu Cys Ala Arg 500 505 510 Thr Val Ser Tyr Asn Gly Ile Leu Gly Pro Glu Cys Gly Thr Lys Tyr 515 520 525 Leu Leu Asn Leu Thr Leu Glu Pro Phe Ser Glu Lys Ser Val Pro Leu 530 535 540 Cys Ile Leu Tyr Glu Lys Tyr Arg Asp Cys Leu Thr Glu Ser Asn Leu 545 550 555 560 Ile Lys Val Arg Ala Leu Leu Val Glu Pro Val lie Asn Ser Tyr Leu 565 570 575 Leu Ala Glu Arg Asp Leu Tyr Leu Glu Asn Pro Glu Ile Lys Ile Arg 580 585 590 Ile Leu Gly Glu Pro Lys Gln Lys Arg Lys Leu Val Ala Glu Val Ser 595 600 605 Leu Gln Asn Pro Leu Pro Val Ala Leu Glu Gly Cys Thr Phe Thr Val 610 615 620 Glu Gly Ala Gly Leu Thr Glu Glu Gln Lys Thr Val Glu Ile Pro Asp 625 630 635 640 Pro Val Glu Ala Gly Glu Glu Val Lys Val Arg Met Asp Leu Val Pro 645 650 655

Leu His Met Gly Leu His Lys Leu Val Val Asn Phe Glu Ser Asp Lys 660 665 670 Leu Lys Ala Val Lys Gly Phe Arg Asn Val Ile Ile Gly Pro Ala 675 680 685 <210> 11 <211> 1470 <212> DNA <213> Homo sapiens <400> 11 gacggtcacc cgttgccagc tctagccttt aaattcccgg ctcggggacc tccacgcacc 60 gcggctagcg ccgacaacca gctagcgtgc aaggcgccgc ggctcagcgc gtaccggcgg 120 gtttcgaaac cgcagtcctc cggcgacccc gaactccgct ccggagcctc agccccctgg 180 aaagtgatcc cggcatcgga gagccaagat gccggcccac ttgctgcagg acgatatctc 240 tagctcctat accaccacca ccaccattac agcgcctcct ccaggggtcc tgcagaatgg 300 aggagataag ttggagacga tgcccctcta cttggaagac gacattcgcc ctgatataaa 360 agatgatata tatgacccca cctacaagga taaggaaggc ccaagcccca aggttgaata 420 tgtctggaga aacatcatcc ttatgtctct gctacacttg ggagccctgt atgggatcac 480 tttgattcct acctgcaagt tctacacctg gctttggggg gtattctact attttgtcag 540 tgccctgggc ataacagcag gagctcatcg tctgtggagc caccgctctt acaaagctcg 600 gctgccccta cggctctttc tgatcattgc caacacaatg gcattccaga atgatgtcta 660 tgaatgggct cgtgaccacc gtgcccacca caagttttca gaaacacatg ctgatcctca 720 taattcccga cgtggctttt tcttctctca cgtgggttgg ctgcttgtgc gcaaacaccc 780 agctgtcaaa gagaagggga gtacgctaga cttgtctgac ctagaagctg agaaactggt 840 gatgttccag aggaggtact acaaacctgg cttgctgatg atgtgcttca tcctgcccac 900 gcttgtgccc tggtatttct ggggtgaaac ttttcaaaac agtgtgttcg ttgccacttt 960 cttgcgatat gctgtggtgc ttaatgccac ctggctggtg aacagtgctg cccacctctt 1020 cggatatcgt ccttatgaca agaacattag cccccgggag aatatcctgg tttcacttgg 1080 agctgtgggt gagggcttcc acaactacca ccactccttt ccctatgact actctgccag 1140 tgagtaccgc tggcacatca acttcaacac attcttcatt gattggatgg ccgccctcgg 1200 tctgacctat gaccggaaga aagtctccaa ggccgccatc ttggccagga ttaaaagaac 1260 cggagatgga aactacaaga gtggctgagt ttggggtccc tcaggttcct ttttcaaaaa 1320 ccagccaggc agaggtttta atgtctgttt attaactact gaataatgct accaggatgc 1380 taaagatgat gatgttaacc cattccagta cagtattctt ttaaaattca aaagtattga 1440 aagccaaaaa aaaaaaaaaa aaaaaaaaaa 1470

<210> 12 <211> 359 <212> PRT <213> Homo sapiens <400> 12 Met Pro Ala His Leu Leu Gln Asp Asp Ile Ser Ser Ser Tyr Thr Thr 1 5 10 15 Thr Thr Thr Ile Thr Ala Pro Pro Pro Gly Val Leu Gln Asn Gly Gly 20 25 30 Asp Lys Leu Glu Thr Met Pro Leu Tyr Leu Glu Asp Asp Ile Arg Pro 35 40 45 Asp Ile Lys Asp Asp Ile Tyr Asp Pro Thr Tyr Lys Asp Lys Glu Gly 50 55 60 Pro Ser Pro Lys Val Glu Tyr Val Trp Arg Asn Ile Ile Leu Met Ser 65 70 75 80 Leu Leu His Leu Gly Ala Leu Tyr Gly Ile Thr Leu Ile Pro Thr Cys 85 90 95 Lys Phe Tyr Thr Trp Leu Trp Gly Val Phe Tyr Tyr Phe Val Ser Ala 100 105 110 Leu Gly Ile Thr Ala Gly Ala His Arg Leu Trp Ser His Arg Ser Tyr 115 120 125 Lys Ala Arg Leu Pro Leu Arg Leu Phe Leu Ile Ile Ala Asn Thr Met 130 135 140 Ala Phe Gln Asn Asp Val Tyr Glu Trp Ala Arg Asp His Arg Ala His 145 150 155 160

His Lys Phe Ser Glu Thr His Ala Asp Pro His Asn Ser Arg Arg Gly 165 170 175 Phe Phe Phe Ser His Val Gly Trp Leu Leu Val Arg Lys His Pro Ala 180 185 190 Val Lys Glu Lys Gly Ser Thr Leu Asp Leu Ser Asp Leu Glu Ala Glu 195 200 205 Lys Leu Val Met Phe Gln Arg Arg Tyr Tyr Lys Pro Gly Leu Leu Met 210 215 220 Met Cys Phe Ile Leu Pro Thr Leu Val Pro Trp Tyr Phe Trp Gly Glu 225 230 235 240 Thr Phe Gln Asn Ser Val Phe Val Ala Thr Phe Leu Arg Tyr Ala Val 245 250 255 Val Leu Asn Ala Thr Trp Leu Val Asn Ser Ala Ala His Leu Phe Gly 260 265 270 Tyr Arg Pro Tyr Asp Lys Asn Ile Ser Pro Arg Glu Asn Ile Leu Val 275 280 285 Ser Leu Gly Ala Val Gly Glu Gly Phe His Asn Tyr His His Ser Phe 290 295 300 Pro Tyr Asp Tyr Ser Ala Ser Glu Tyr Arg Trp His Ile Asn Phe Asn 305 310 315 320 Thr Phe Phe Ile Asp Trp Met Ala Ala Leu Gly Leu Thr Tyr Asp Arg 325 330 335 Lys Lys Val Ser Lys Ala Ala Ile Leu Ala Arg Ile Lys Arg Thr Gly 340 345 350

Asp Gly Asn Tyr Lys Ser Gly 355 <210> 13 <211>1637 <212> DNA <213> Homo sapiens <400> 13 gaggcgaacc ggagcgcggg gccgcggtcg ccccgaccag agccgggaga ccgcagcacc 60 cgcagccgcc cgcgagcgcg ccgaagacag cgcgcaggcg agagcgcgcg ggcgggggcg 120 <BR> <BR> <BR> cgcaggccct gcccgcccct tccgtcccca cccccctccg ccctttcctc tccccacctt 180 cctctcgcct cccgcgcccc cgcaccgggc gcccaccctg tcctcctcct gcgggagcgt 240 tgtccgtgtt ggcggccgca gcgggccggg ccggtccggc gggccggggg atggcgctgc 300 tggacctggc cttggaggga atggccgtct tcgggttcgt cctcttcttg gtgctgtggc 360 tgatgcattt catggctatc atctacaccc gattacacct caacaagaag gcaactgaca 420 aacagcctta tagcaagctc ccaggtgtct ctcttctgaa accactgaaa ggggtagatc 480 ctaacttaat caacaacctg gaaacattct ttgaattgga ttatcccaaa tatgaagtgc 540 tcctttgtgt acaagatcat gatgatccag ccattgatgt atgtaagaag cttcttggaa 600 aatatccaaa tgttgatgct agattgttta taggtggtaa aaaagttggc attaatccta 660 aaattaataa tttaatgcca ggatatgaag ttgcaaagta tgatcttata tggatttgtg 720 atagtggaat aagagtaatt ccagatacgc ttactgacat ggtgaatcaa atgacagaaa 780 aagtaggctt ggttcacggg ctgccttacg tagcagacag acagggcttt gctgccacct 840 tagagcaggt atattttgga acttcacatc caagatacta tatctctgcc aatgtaactg 900 gtttcaaatg tgtgacagga atgtcttgtt taatgagaaa agatgtgttg gatcaagcag 960 gaggacttat agcttttgct cagtacattg ccgaagatta ctttatggcc aaagcgatag 1020 ctgaccgagg ttggaggttt gcaatgtcca ctcaagttgc aatgcaaaac tctggctcat 1080 attcaatttc tcagtttcaa tccagaatga tcaggtggac caaactacga attaacatgc 1140 ttcctgctac aataatttgt gagccaattt cagaatgctt tgttgccagt ttaattattg 1200 gatgggcagc ccaccatgtg ttcagatggg atattatggt atttttcatg tgtcattgcc 1260 tggcatggtt tatatttgac tacattcaac tcaggggtgt ccagggtggc acactgtgtt 1320 tttcaaaact tgattatgca gtcgcctggt tcatccgcga atccatgaca atatacattt 1380 ttttgtctgc attatgggac ccaactataa gctggagaac tggtcgctac agattacgct 1440 gtgggggtac agcagaggaa atcctagatg tataactaca gctttgtgac tgtatataaa 1500 ggaaaaaaga gaagtattat aaattatgtt tatataaatg cttttaaaaa tctaccttct 1560 gtagttttat cacatgtatg ttttggtatc tgttctttaa tttatttttg catggcactt 1620 gcatctgtga aaaaaaa 1637

<210> 14 <211> 394 <212> PRT <213> Homo sapiens <400> 14 Met Ala Leu Leu Asp Leu Ala Leu Glu Gly Met Ala Val Phe Gly Phe 1 5 10 15 Val Leu Phe Leu Val Leu Trp Leu Met His Phe Met Ala Ile Ile Tyr 20 25 30 Thr Arg Leu His Leu Asn Lys Lys Ala Thr Asp Lys Gln Pro Tyr Ser 35 40 45 Lys Leu Pro Gly Val Ser Leu Leu Lys Pro Leu Lys Gly Val Asp Pro 50 55 60 Asn Leu Ile Asn Asn Leu Glu Thr Phe Phe Glu Leu Asp Tyr Pro Lys 65 70 75 80 Tyr Glu Val Leu Leu Cys Val Gln Asp His Asp Asp Pro Ala Ile Asp 85 90 95 Val Cys Lys Lys Leu Leu Gly Lys Tyr Pro Asn Val Asp Ala Arg Leu 100 105 110 Phe Ile Gly Gly Lys Lys Val Gly Ile Asn Pro Lys Ile Asn Asn Leu 115 120 125 Met Pro Gly Tyr Glu Val Ala Lys Tyr Asp Leu Ile Trp Ile Cys Asp 130 135 140 Ser Gly Ile Arg Val Ile Pro Asp Thr Leu Thr Asp Met Val Asn Gln 145 150 155 160

Met Thr Glu Lys Val Gly Leu Val His Gly Leu Pro Tyr Val Ala Asp 165 170 175 Arg Gln Gly Phe Ala Ala Thr Leu Glu Gln Val Tyr Phe Gly Thr Ser 180 185 190 His Pro Arg Tyr Tyr Ile Ser Ala Asn Val Thr Gly Phe Lys Cys Val 195 200 205 Thr Gly Met Ser Cys Leu Met Arg Lys Asp Val Leu Asp Gln Ala Gly 210 215 220 Gly Leu Ile Ala Phe Ala Gln Tyr Ile Ala Glu Asp Tyr Phe Met Ala 225 230 235 240 Lys Ala Ile Ala Asp Arg Gly Trp Arg Phe Ala Met Ser Thr Gln Val 245 250 255 Ala Met Gln Asn Ser Gly Ser Tyr Ser Ile Ser Gln Phe Gln Ser Arg 260 265 270 Met Ile Arg Trp Thr Lys Leu Arg Ile Asn Met Leu Pro Ala Thr Ile 275 280 285 Ile Cys Glu Pro Ile Ser Glu Cys Phe Val Ala Ser Leu Ile Ile Gly 290 295 300 Trp Ala Ala His His Val Phe Arg Trp Asp Ile Met Val Phe Phe Met 305 310 315 320 Cys His Cys Leu Ala Trp Phe Ile Phe Asp Tyr Ile Gln Leu Arg Gly 325 330 335 Val Gln Gly Gly Thr Leu Cys Phe Ser Lys Leu Asp Tyr Ala Val Ala 340 345 350

Trp Phe Ile Arg Glu Ser Met Thr Ile Tyr Ile Phe Leu Ser Ala Leu 355 360 365 Trp Asp Pro Thr Ile Ser Trp Arg Thr Gly Arg Tyr Arg Leu Arg Cys 370 375 380 Gly Gly Thr Ala Glu Glu Ile Leu Asp Val 385 390 <210>15 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400>15 ggccagtgaa ttgtaatacg actcactata gggaggcggt tttttttttt tttttttttt 60 ttt 63 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 16 gtcgtcaaga tgctaccgtt cagga 25 <210> 17 <211> 51

<212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 17 ggggacaagt ttgtacaaaa aagcaggcta tgccgatgtt catcgtaaac a 51 <210> 18 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 18 ggggaccact ttgtacaaga aagctgggtt taggcgaagg tggagttgtt 50 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 19 aaggattcgg gaatgggctg tcagaccaga ct 32 <210> 20 <211> 31 <212> DNA

<213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 20 ttaagctttc atcttttctt tttctgttgc c 31