CN1636070A - Nucleic acid encoding a G-protein-coupled receptor, and uses thereof - Google Patents

Abstract

Provided herein is a novel and useful G-protein coupled receptor that is involved in signal transduction with respect to inflammation and physiological immunological response. Also provided are methods of using the receptor to screen for molecules that may modulate the activity of the receptor. Such molecules may readily have applications in treating a plethora of inflammation and immunological related diseases and disorders.

Description

Nucleic acid encoding a G protein-coupled receptor and use thereof

field of invention

The present invention generally relates to a novel nucleic acid molecule encoding a currently unknown G protein-coupled receptor GAVE18, and to uses of the nucleic acid molecule and GAVE18.

Background of the invention

G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins involved in cellular signal transduction. GPCRs respond to a variety of extracellular signals including neurotransmitters, hormones, odorants and light, and can transduce signals to initiate intracellular second messenger responses. Many therapeutic agents target GPCRs because these receptors mediate a variety of physiological responses, including inflammation, vasodilation, heart rate, bronchodilation, endocrine and peristalsis.

GPCRs are characterized by having an extracellular domain, seven transmembrane domains, and an intracellular domain. Some functions performed by such receptors, such as binding ligands and interacting with G proteins, are related to the presence of certain amino acids at key positions. For example, many studies have shown that differences in amino acid sequence in GPCRs lead to differences in affinity with natural ligands or small molecule agonists or antagonists. In other words, small differences in sequence can result in different binding affinities and activities. (seeing as people such as Meng, J Biochem (1996) 271 (50): 32016-20; Burd et al, J Bio Chem (1998) 273 (51): 34488-95 and Hurley et al., J Neurochem (1999) 72(1):413-21). Specifically, studies have shown that differences in the amino acid sequence in the third intracellular domain can result in different activities. Myburgh et al found that alanine 261 in the 3rd intracellular loop of the gonadotropin-releasing hormone receptor is critical for G protein coupling and receptor internalization (Biochem J (1998) 331(p. 3 parts): 893-6). Wonerow et al. studied the thyrotropin receptor and demonstrated that genetic deletion in the third intracellular loop results in constitutive receptor activity (J Bio Chem (1998) 273(14):7900-5).

In general, the binding of an endogenous ligand to a receptor results in a conformational change in the intracellular domain of the receptor, enabling coupling between the intracellular domain and the intracellular component G protein. Several G proteins exist, such as _Gq , _Gs , _Gi , _Gz , and _Go (see eg, Dessauer et al., Clin Sci (Colch) (1996) 91(5):527-37). The third intracellular loop (IC-3 loop) and the carboxyl terminus of the receptor interact with the G protein (Pauwels et al., Mol Neurobiol (1998) 17(1-3): 109-135 and Wonerow et al, see above). Some GPCRs are "pan-primarily binding" with respect to G proteins, ie, one GPCR can interact with more than one G protein (see, eg, Kenakin, Life Sciences (1988) 43:1095).

Coupling of ligand-activated GPCRs to G proteins initiates a signaling cascade process (termed "signal transduction"). Such signal transduction ultimately leads to cellular activation or cellular inhibition.

GPCRs in the cell membrane exist in equilibrium in two different conformations: an "inactive" state and an "active" state. Receptors in an inactive state cannot be linked to intracellular signal transduction pathways to produce a biological response (exceptions exist, such as when receptors are overexpressed in transduced cells, see e.g. www.creighton.edu/Pharmacology/inverse.htm .) . The conformational transition to the active state allows the receptor to connect to the transduction pathway (via the G protein) and generate a biological response. An agonist binds and makes the active conformation more likely. Sometimes, however, such receptors are said to be constitutively active (ie already in an active conformation or ligand-independent or spontaneously active state) if there has been a substantial response in the absence of any agonist. When agonists are added to such systems, enhanced responses are often observed. However, when classical antagonists are added, the binding of such molecules has no effect on the receptor. On the other hand, certain antagonists can cause inhibition of the constitutive activity of the receptor, suggesting that the latter class of drugs is not technically an antagonist, but an agonist with the opposite intrinsic activity. These drugs are called inverse agonists ( www.creighton.edu/Pharmacology/inverse.htm.) .

Conventional research on receptors is based on the assumption of starting with the identification of endogenous ligands and then moving forward to identify antagonists and other receptor effector molecules. Even where antagonists are discovered first, the dogmatic response is to identify the endogenous ligand (WO 00/22131). But since the active state is most useful for testing screening purposes, access to such constitutive receptors and especially constitutive GPCRs will allow easy separation of agonists, partial agonists, inverse agonists and antagonists in the absence of endogenous ligand information agent. Also, for diseases due to disturbances in receptor activity, drugs that cause constitutive inhibition of activity, or more specifically drugs that reduce the concentration of effectively activated receptors, can more easily be found by testing using receptors in a spontaneously activated state. For example as receptors that can be transfected into a patient for the treatment of disease, the activity of such receptors can be fine-tuned with inverse agonists discovered by the assays described above.

Diseases such as asthma, chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis (RA) are generally believed to have an inflammatory etiology involving T helper cells, monocyte-macrophages and eosinophils. Current anti-inflammatory treatments with corticosteroids are effective for asthma but are associated with metabolic and endocrine side effects. The same problem may arise with inhaled formulations that are absorbed through the lung or nasal mucous membranes. There is currently no satisfactory oral therapy for RA or COPD.

Eosinophils mediate most of the airway disturbances in allergy and asthma. Interleukin-5 (IL-5) is a cytokine for the growth and activation of eosinophils. Studies have shown that IL-5 is necessary for tissue eosinophilia and airway overreaction to eosinophil-mediated tissue damage (Chang et al., J Allergy Clin Immunol (1996) 98(5pt 1): 922 -931 and Duez et al., Am J Respir Crit Care Med (2000) 161(1):200-206). In atopic asthma, T-helper-2 (Th2) cells produce IL-5 upon exposure to an allergen (eg, house dust mite antigen).

Rheumatoid arthritis is thought to result from the accumulation of activated macrophages in the affected synovium. Interferon gamma (IFNγ) is a cytokine secreted by T-helper-1 (Th1) cells with many pro-inflammatory properties. It is the most potent macrophage-activating cytokine and induces MHC class II gene transcription leading to a dendritic cell-like phenotype.

Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria that causes an inflammatory response, including the release of tumor necrosis factor alpha (TNFα). The efficacy of intravenous anti-TNFα in the treatment of rheumatoid arthritis has been clinically proven. COPD is also thought to be caused by the accumulation of macrophages in the lungs, which produce chemoattractants for neutrophils (e.g. IL-8: de Boer et al., J Pathol (2000) 190(5) : 619-626). Both macrophages and neutrophils release cathepsins that cause degradation of alveolar walls. Lung epithelial cells are considered to be an important source of inflammatory cell chemoattractants and other inflammatory cell activators (see, e.g., Thomas et al., J Virol (2000) 74(18): 8425-8433; Lamkhioued et al., Am J Respir Crit Care Med (2000) 162(2Pt. 1): 723-732; and Sekiya et al., J Immunol (2000) 165(4): 2205-2213).

Given that GPCRs play a role in disease and can treat disease by modulating the activity of GPCRs, the identification and characterization of previously unknown GPCRs may allow the development of new compositions and methods for the treatment of diseases involving GPCR activity. Therefore, what is needed is the discovery, isolation and characterization of novel and useful nucleic acid molecules encoding hitherto unknown GPCRs.

There is also a need for assays that utilize such currently unknown GPCRs to identify molecules that may act as potential agonists or antagonists of particular GPCRs. These molecules may serve as therapeutic agents that modulate the activity of GPCRs in vivo, and thereby, treat the plethora of diseases associated with GPCR activity.

The citation of any reference herein shall not be construed as an admission that such reference is available as prior art to the present application.

Summary of the invention

The present invention identifies and describes the expression of a novel constitutively active GPCR, GAVE18, and provides compositions and methods for using this discovery to identify and treat related diseases.

Broadly, therefore, the invention extends to isolated nucleic acid molecules comprising the DNA sequence of Figure 5 (SEQ ID NO: 1), variants, fragments, analogs or derivatives thereof. Such variants of the invention may be allelic variants, degenerate variants or allelic variants that result in altered sequence degeneracy.

Further the invention extends to an isolated nucleic acid molecule capable of hybridizing to an isolated nucleic acid molecule of SEQ ID NO: 1 or a variant thereof under stringent hybridization conditions. Still further, the present invention extends to an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule that is complementary to the DNA sequence of SEQ ID NO:1. Stringent hybridization conditions are described below.

Further, the present invention extends to an isolated nucleic acid molecule comprising a DNA sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

Optionally, the isolated nucleic acid molecules of the invention described above may be detectably labeled. Examples of detectable labels for use herein include, but are not limited to, enzymes, radioisotopes, or chemiluminescent substances. Examples of specific detectable labels are described below.

The invention also encompasses specific polypeptides. For example, the invention extends to purified polypeptides comprising the amino acid sequence of SEQ ID NO: 2, conservative variants thereof, analogs or derivatives thereof. Optionally, polypeptides of the invention may be detectably labeled.

Furthermore, the invention extends to antibodies, wherein the polypeptide of the invention is the immunogen used to generate the antibody. Antibodies can be monoclonal or polyclonal. Further, antibodies may be "chimeric", eg, they may comprise protein domains of antibodies raised against purified polypeptides of the invention in a different species of animal. In a specific embodiment, an antibody of the invention may be "humanized". Antibodies of the invention may, of course, be detectably labeled. Examples of specific detectable labels for use herein are described below.

The present invention further extends to an expression vector comprising a nucleic acid molecule operably linked to an expression control element, wherein said nucleic acid molecule comprises the DNA sequence of SEQ ID NO: 1, a variant, analog or derivative thereof, or a fragment thereof. Further, the expression vector of the present invention can comprise such an isolated nucleic acid molecule, which can hybridize under stringent hybridization conditions with an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1 operably linked to expression control elements, or can be hybridized in Hybridization under stringent hybridization conditions to a hybridization probe complementary to an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1, wherein the hybridization probe is operably linked to a nucleic acid expressing a regulatory element. A specific example of an expression control element that has been used herein is a promoter. Examples of specific promoters applicable to the present invention include, but are not limited to, the early promoter of hCMV, the early promoter of SV40, the early promoter of adenovirus, the early promoter of vaccinia virus, the early promoter of polyoma virus, the SV40 Late promoter of adenovirus, late promoter of adenovirus, late promoter of vaccinia virus, late promoter of polyomavirus, lac system, trp system, TAC system, TRC system, the main operator gene and promoter region of lambda phage, Regulatory region of fd coat protein, 3-phosphoglycerol kinase promoter, acid phosphatase promoter or yeast alpha mating factor promoter.

The host cell can be transfected or transformed with the expression vector of the present invention, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof, can be produced. Host cells can be prokaryotic or eukaryotic. Specific examples of unicellular hosts that have been used here include E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1 , B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells, only some of which are mentioned here.

Further, the present invention also extends to methods for producing such a purified polypeptide, wherein said purified polypeptide comprises the amino acid sequence of SEQ ID NO: 2, a variant thereof or a fragment thereof. Such methods involve culturing a host cell transformed or transfected with an expression vector of the invention under conditions that provide expression of the purified polypeptide and then recovering the purified polypeptide from the unicellular host, the culture surrounding the host cell, or both.

Further, the invention extends to assays for identifying compounds that modulate GAVE18 activity. Such compounds may be agonists, antagonists or inverse agonists of GAVE18. Accordingly, the invention extends to a method for determining an agonist of GAVE18 comprising contacting a potential agonist with a cell expressing GAVE18 in the presence of an endogenous ligand, and determining when the potential agonist is present , whether the signaling activity of GAVE18 is enhanced relative to the signaling activity of GAVE18 in the absence of the potential agonist.

Also, the invention extends to methods for identifying inverse agonists of GAVE18. The method involves contacting a potential inverse agonist with cells expressing GAVE18 and determining whether the signaling activity of GAVE18 is relative to the presence of endogenous inverse agonist in the presence of the potential inverse agonist and an endogenous ligand or agonist The signaling activity of GAVE18 is reduced in the absence of ligand or agonist but in the absence of this potential inverse agonist, and is also reduced in the presence of endogenous ligand or agonist.

Naturally, the present invention extends to a method for identifying antagonists of GAVE18 comprising contacting a potential antagonist with cells expressing GAVE18, and determining whether the signaling activity of GAVE18 in the presence of said potential antagonist is relative to the presence of GAVE18 activity is reduced in response to endogenous ligands or agonists.

Accordingly, it is an object of the present invention to provide isolated nucleic acid sequences encoding GAVE18 protein, fragments thereof or variants thereof.

Another object of the present invention is to provide nucleic acid molecule variants comprising the DNA sequence of SEQ ID NO: 1, or nucleic acid molecule variants that can be hybridized with SEQ ID NO: 1 under stringent hybridization conditions.

Yet another object of the present invention is to provide the amino acid sequence of GAVE18, its variants, fragments, analogs or derivatives.

Yet another object of the present invention is to provide an expression vector comprising a DNA sequence encoding GAVE18, a variant, fragment, analog or derivative thereof, operably linked to an expression regulatory element.

Yet another object of the present invention is to provide antibodies produced using GAVE18, its variants, analogs or derivatives, or fragments thereof as an immunogen.

Another object of the present invention relates to methods for identifying compounds that modulate the activity of GAVE18. Such modulators may be antagonists of GAVE18, agonists of GAVE18 or inverse agonists of GAVE18.

Another object of the present invention is to provide a pharmaceutical composition for modulating the activity of GAVE18. Such modulation can be used to treat a variety of diseases associated with GAVE18 activity, eg, various inflammatory diseases, asthma, chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis, to name a few.

These and other aspects of the invention will be better understood by reference to the following detailed description and accompanying drawings.

Brief description of the drawings

Figure 1 : Northern blot showing transcription of GAVE18 DNA in different types of tissues, especially "immunologically relevant" tissues such as thymus and liver.

Figure 2: Bar graph showing relative expression of GAVE18 DNA in different cell types.

Figure 3: Histogram showing the relative expression of GAVE18 DNA in different types of tissues.

Figure 4: GAVE18 expression profile. The data in this expression profile shows that the expression level of GAVE18 is increased in NHBE (normal human bronchial epithelial cells) treated with tumor necrosis factor alpha (TNFα). TNF[alpha] is always seen in inflamed tissue. In addition, bronchial epithelial cells play an important role in asthma.

Figure 5: DNA sequence of GAVE 18 (SEQ ID NO: 1).

Figure 6: Deduced GAVE18 amino acid sequence (SEQ ID NO: 2).

Detailed description of the invention

As explained above, the present invention involves the surprising and unexpected discovery of a hitherto unknown nucleic acid molecule encoding a hitherto unknown G protein coupled receptor referred to herein as GAVE18. Specifically, GAVE18 was found to be expressed in immune tissues or organs such as kidney, liver and small intestine. Furthermore, it was surprisingly and unexpectedly found that the expression level of GAVE18 was increased in NHBE (normal human bronchial epithelial cells) treated with tumor necrosis factor alpha (TNFα), which is always seen in inflamed tissues a protein. Therefore, altering the activity of GAVE18 may be useful in the treatment of immune-related diseases and disorders, such as various inflammatory diseases and rheumatoid arthritis.

Various terms and phrases used in this specification and claims to describe the present invention are as follows:

As used herein, the term "modulator" refers to a molecule (such as, but not limited to, ligands and candidate compounds) that modulates the activity of GAVE18. Modulators of the invention may be agonists, partial agonists, antagonists or inverse agonists of GAVE18.

The term "agonist" as used herein refers to a molecule (such as, but not limited to, ligands and candidate compounds) that activates an intracellular response or enhances the binding of GTP to a membrane when bound to a receptor.

As used herein, the term "partial agonist" refers to a molecule that, when bound to a receptor, activates an intracellular response to a lesser extent or enhances the binding of GTP to a membrane to a lesser extent than an agonist ( such as, but not limited to, ligands and candidate compounds).

The term "antagonist" as used herein refers to a molecule (such as, but not limited to, ligands and candidate compounds) that competes for binding to a receptor at the same site as an agonist. Antagonists, however, do not activate intracellular responses initiated by the active form of the receptor, and thus may inhibit intracellular responses elicited by agonists or partial agonists. In a related aspect, an antagonist does not reduce baseline intracellular responses when no agonist or partial agonist is present.

The term "inverse agonist" as used herein refers to a molecule (such as, but not limited to, ligands and candidate compounds) that binds to a constitutively activated receptor and inhibits a baseline intracellular response. A baseline response is initiated by the active form of the receptor below the normal basal level of activity observed in the absence of an agonist or partial agonist or reduced GTP binding to the membrane.

As used herein, the term "candidate compound" refers to a molecule (such as, but not limited to, a chemical compound) that is amenable to screening techniques. In one embodiment, the term excludes known compounds selected from agonists, partial agonists, inverse agonists or antagonists of GAVE18. Those compounds are identified by traditional drug development methods involving identification of receptor-specific endogenous ligands and/or screening of candidate compounds that antagonize the receptor, where such screening requires competition assays to assess efficacy.

The terms "constitutively activated receptor" or "spontaneously activated receptor" are used herein interchangeably and refer to a receptor that is activated in the absence of a ligand. Such constitutively activated receptors may be endogenous (such as GAVE18) or non-endogenous; GPCRs may be modified by recombinant means to generate mutant constitutive forms of wild-type GPCRs (see e.g. EP 1071701; WO 00/22129; WO 00/22131; and U.S. Patent Nos. 6,150,393 and 6,140,509, incorporated herein by reference).

The term "constitutive receptor activation" as used herein refers to the stabilization of a receptor in an active state by means other than the binding of the receptor to an endogenous ligand or its chemical equivalent.

The term "ligand" as used herein refers to a molecule that binds another molecule, such as, but not limited to, a hormone or a neurotransmitter, and further wherein the molecule stereoselectively binds to receptor binding.

The term "family" when referring to the proteins and nucleic acid molecules of the present invention refers to two or more proteins or nucleic acid molecules having an apparent common domain and having sufficient amino acid or nucleotide sequence identity as defined herein . Such family members may occur naturally and may be from the same or different species. For example, a family may comprise a first protein of human origin and a homologue of that protein of murine origin, and a second, different protein of human origin and a murine homolog of the second protein. Family members may also share functional characteristics.

"GAVE18 activity", "GAVE18 biological activity" or "GAVE18 functional activity" interchangeable herein refers to the activity of GAVE18 protein, polypeptide or nucleic acid molecule on GAVE18 effector cells measured in vivo or in vitro according to standard techniques. active. The GAVE18 activity may be a direct activity or an indirect activity, wherein the direct activity is such as a binding activity to a second protein or an enzymatic activity to a second protein, wherein the indirect activity is such as mediated by the interaction of the GAVE18 protein with the second protein Cell signaling activity. In a preferred embodiment, GAVE18 activity comprises at least one or more of the following activities: (i) the ability to interact with proteins in the GAVE18 signaling pathway; (ii) the ability to interact with GAVE18 ligands; and (iii) ) ability to interact with intracellular target proteins.

Furthermore, conventional molecular biology, microbiology and recombinant DNA techniques in the art can be used according to the present invention. Such techniques are fully described in the references listed below. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al., 1989″); DNA Cloning: A Practical Approach (DNA Cloning: A Practical Approach), Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (Nucleic Acid Hybridization) Hybridization) [Editors B.D.Hames & S.J.Higgins (1985)]; Transcription And Translation [Editors B.D.Hames & S.J.Higgins (1984)]; Animal Cell Culture [Editors R.I.Freshney (1986)]; Fixed Immobilized CellsAnd Enzymes [IRL Press, (1986)]; B.Perbal, A Practical Guide To Molecular Cloning (1984); F.M.Ausubel et al. (eds), Molecular Biology Current Methods (Current Protocols in Molecular Biology), John Wiley & Sons, Inc. (1994).

Accordingly, if appearing in the text, the following terms have the definitions given below.

A "vector" is a replicon, such as a plasmid, phage or cosmid, to name only a few, to which another DNA segment can be ligated thereby causing replication of the ligated DNA segment. A "replicon" is any genetic element (eg, plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, ie, replicates under its own control. Specific examples of carriers are described below.

A "cassette" refers to a DNA segment that can be inserted into a vector at specific restriction sites. The DNA fragment encodes the polypeptide of interest, and the cassette and restriction sites are designed to ensure that the cassette is inserted in the correct reading frame for transcription and translation.

A cell is "transfected" with exogenous or heterologous DNA when the exogenous or heterologous DNA has been introduced into the cell. A cell is "transformed" with exogenous or heterologous DNA when transfection of that DNA results in a change in the cell's phenotype. Preferably, the transforming DNA is integrated (covalently linked) into the chromosomal DNA that makes up the genome of the cell.

"Heterologous"DNA refers to DNA that is not naturally present in the cell or chromosomal locus of the cell. Preferably, the heterologous DNA includes genes foreign to the cell.

"Homologous recombination" refers to the insertion of a vector's foreign DNA sequence into a chromosome. Specifically, the vector targets specific chromosomal loci for homologous recombination. For specific homologous recombination, the vector contains regions of homology to chromosomal sequences of sufficient length to allow complementary binding and chromosomal incorporation of the vector. Longer regions of homology and higher degrees of sequence similarity increase the efficiency of homologous recombination.

Isolated nucleic acid molecules of the invention

In one aspect, the invention extends to an isolated nucleic acid molecule comprising the DNA sequence of Figure 5 (SEQ ID NO: 1), variants, fragments, analogs or derivatives thereof.

"Nucleic acid molecule" means a ribonucleoside (adenosine, guanosine, uridine, or cytosine; an "RNA molecule") or a deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxythymine, or deoxycytosine;" DNA molecule"), or any of its phosphate analogs, such as phosphorothioate and thioester, in single-stranded or double-helix form. Can be double stranded DNA-DNA, DNA-RNA and RNA-RNA helices. The term nucleic acid molecule, specifically a DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular form of tertiary structure. Thus, the term includes double-stranded DNA (eg, restriction fragments), plasmids, and chromosomes, present as linear DNA molecules or circular DNA molecules, and the like. In discussing the structure of a particular double-stranded DNA molecule, sequence is described herein as the sequence along the 5' to 3' direction of the untranscribed strand of DNA (i.e., the sequence of that strand is homologous to mRNA), according to conventional convention. A "recombinant DNA molecule" is a DNA molecule that has been manipulated by molecular biology.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that exist in the natural source of the nucleic acid. In particular, an "isolated" nucleic acid is free of the sequences naturally flanking the GAVE18-encoding nucleic acid (ie, sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. In various embodiments, an isolated GAVE18 nucleic acid molecule can comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nuclear DNA that flanks the nucleic acid molecule naturally in the genomic DNA of the cell from which the nucleic acid is derived. nucleotide sequence. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or can be substantially free of other cellular material or culture medium when produced by chemical synthesis. Contains no chemical precursors or other chemicals.

Nucleic acid molecules of the present invention, such as nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1 or any fragment or complementary fragment thereof, or analogs or derivatives thereof, can be applied standard molecular biology techniques and the sequence information provided here for isolation. GAVE18 nucleic acid molecules can be isolated by standard hybridization and cloning techniques (as described by Sambrook et al.) using all or part of the nucleic acid sequence of SEQ ID NO: 1 as a hybridization probe.

A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and using appropriate oligonucleotide primers according to standard PCR amplification techniques. Such primers are readily prepared using the information set forth in SEQ ID NO: 1 and routine experimental techniques. The amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to the GAVE18 nucleotide sequence can be prepared by standard synthetic techniques, such as by an automated DNA synthesizer.

Isolated nucleic acid molecule hybridizable to GAVE18 DNA

The invention further extends to isolated nucleic acid molecules that hybridize to GAVE18 DNA, to hybridization probes that are complementary to GAVE18 DNA under stringent hybridization conditions, or to both under stringent hybridization conditions. In particular, the present invention extends to an isolated nucleic acid molecule which, under stringent hybridization conditions, hybridizes to a nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1, or to a probe which is complementary to a DNA sequence comprising An isolated nucleic acid molecule of the DNA sequence of SEQ ID NO:1.

A nucleic acid molecule can hybridize to another nucleic acid molecule when its single-stranded form can anneal to another nucleic acid molecule, such as cDNA, genomic DNA, or RNA, under suitable conditions of temperature and solution ionic strength (see Sambrook et al., see above). Temperature and ionic strength determine the "stringency" of hybridization. For initial screening of homologous nucleic acids, low stringency hybridization conditions corresponding to Tm55°C can be used, such as 5x SSC, 0.1% SDS, 0.25% milk and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderately stringent hybridization conditions correspond to higher Tm, such as 40% formamide, 5x or 6x SSC. Highly stringent hybridization conditions correspond to the highest Tm, such as 50% formamide, 5x or 6x SSC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate level of stringency for hybridization of nucleic acids depends on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the similarity or homology between two nucleotide sequences, the greater the Tm at which nucleic acids having those sequences will hybridize. The relative stability of nucleic acid hybridization (corresponding to higher Tm) decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybridizations longer than 100 nucleotides, there is an equation for calculating the Tm (see Sambrook et al., supra, 9.50-0.51). For hybridization to shorter nucleic acids, ie, oligonucleotides, the location of the mismatch becomes important, and the length of the oligonucleotide determines the specificity of the hybridization (see Sambrook et al., supra, 11.7-11.8). The minimum length of a hybridizable nucleic acid molecule is at least about 20 nucleotides; specifically at least about 30 nucleotides; more specifically at least about 40 nucleotides, even more specifically at least about 50 nucleotides, and More specifically at least about 60 nucleotides. In a particular embodiment of the invention, the hybridizable nucleic acid molecule of the invention is at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000 or 1100 nucleotides in length, and hybridize under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, preferably its coding sequence, its complementary fragment sequence, or its fragment.

The term "hybridizes under stringent conditions" as used herein is a description of hybridization and washing conditions under which nucleotide sequences are at least 55%, 60%, 65%, 70% and preferably 75% or higher complementarity generally remains hybridized. Such stringent conditions are well known to those skilled in the art and can be found in "Current Protocols in Molecular Biology", John Wiley & Sons, N.Y (1989), 6.3.1-6.3.6 turn up. A preferred non-limiting example of stringent hybridization conditions is: hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by washing in 0.2X SSC, 0.1% SDS at 50-65°C one or more times. Preferably, an isolated nucleic acid molecule of the invention which hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 or its complement corresponds to a naturally occurring nucleic acid molecule. A "naturally occurring" nucleic acid molecule as used herein refers to an RNA or DNA molecule having a nucleotide sequence as found in nature (eg, encoding a native protein). The skilled artisan will understand that conditions can be modified for sequence-specific variables (eg, length, G-C abundance, etc.).

The present invention contemplates encompassing GAVE18 nucleic acid fragments that are useful in the diagnosis of GAVE18-like molecules with similar properties. Diagnostic fragments can be derived from any portion of the GAVE18 gene, including flanking sequences. This fragment can be used as a library probe using a known method.

Furthermore, the nucleic acid molecule of the present invention may comprise only a portion of the nucleic acid sequence encoding GAVE18, such as a fragment useful as a probe or primer or a fragment encoding a biologically active portion of GAVE18. For example, such a fragment may include, but is not limited to, the region encoding amino acid residues from about 1 to about 14 of SEQ ID NO:2. The nucleotide sequence determined from cloning the human GAVE18 gene allows the generation of probes and primers for the identification and/or cloning of GAVE18 homologs in other cell types (e.g., from other tissues) as well as GAVE18 homologs from other mammals . Probes/primers generally comprise substantially purified oligonucleotides. Oligonucleotides generally comprise a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 of the sense or antisense sequences of SEQ ID NO: 1 or natural mutants of SEQ ID NO: 1 , preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides. Probes based on the nucleotide sequence of human GAVE18 can be used to detect transcripts or genomic sequences encoding similar or identical proteins.

The term "fragment" or "portion" of an isolated nucleic acid molecule of the invention as used herein comprises at least 12, in particular about 25, more in particular about 50, 75, 100, 125, 150, 175 , 200, 250, 300, 350 or 400 consecutive nucleotides. Thus, a "fragment" of an isolated nucleic acid molecule of the invention is not just 1 or 2 nucleotides.

Likewise, a "fragment" or "portion" of a polypeptide of the invention comprises at least 9 contiguous amino acid residues. A specific example of a polypeptide fragment of the invention comprises an epitope that binds to a GAVE18 antibody or fragment thereof.

The nucleic acid fragment encoding "the biologically active part of GAVE18" can be obtained by isolating the part of SEQ ID NO: 1 encoding a polypeptide having GAVE18 biological activity, expressing the coding part of the GAVE18 protein (such as in vitro recombinant expression) and evaluating the coding part of GAVE18 prepared for its activity. The present invention also includes nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO: 1 due to the degeneracy of the genetic code and thus encode the same GAVE18 protein as the nucleotide sequence shown in SEQ ID NO: 1.

homologous nucleic acid molecules

The present invention also extends to such isolated nucleic acid molecule, it is homologous to GAVE18 DNA molecule, for example it is homologous to the isolated nucleic acid molecule that has SEQ ID NO:1 DNA sequence. Two DNA sequences are "substantially homologous" or "substantially homologous" when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over a given DNA sequence length substantially similar". Substantially homologous sequences can be determined by comparing sequences using standard software available in sequence databases, using default parameters, or by Southern hybridization experiments under stringent hybridization conditions, eg, established for that particular system. The formulation of appropriate hybridization conditions is within the skill of the art. See, eg, Maniatis et al., supra; DNA Cloning, Volumes I and II, supra; Nucleic Acid Hybridization, supra. Furthermore, nucleic acid molecules encoding GAVE18 proteins from other species and having a nucleotide sequence different from human GAVE18 (GAVE18 homologues) are also included in the scope of the present invention.

Variants of the Isolated Nucleic Acid Molecules of the Invention

The present invention also extends to variants of such an isolated nucleic acid molecule, wherein said isolated nucleic acid molecule comprises the DNA sequence of SEQ ID NO:1. Such variants may be degenerate variants, allelic variants or combinations thereof.

Nucleic acid molecules corresponding to natural allelic variants and homologues of the GAVE18 cDNA of the present invention can be based on identity with the human GAVE18 nucleic acid disclosed herein, using the human cDNA or a portion thereof as a hybridization probe, according to standard hybridization techniques in isolated under stringent hybridization conditions.

As used herein, the term "corresponds to" refers to a similar or homologous sequence, whether its exact position is the same or different from the molecule with which the similarity or homology is determined. Thus, the term "corresponds to" refers to sequence similarity, rather than the numbering of amino acid residues or nucleotide bases.

Moreover, due to the degeneracy of the codons of the genetic code, the GAVE18 protein of the present invention can be encoded by many isolated nucleic acid molecules. "Degeneracy signature" refers to the use of different three-letter codes to designate specific amino acids according to the genetic code. The following codons are known in the art to be used interchangeably to encode each particular amino acid:

Phenylalanine (Phe or F) UUU or UUC

Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG

Isoleucine (Ile or I) AUU or AUC or AUA

Methionine (Met or M) AUG

Valine (Val or V) GUU or GUC or GUA or GUG

Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC

Proline (Pro or P) CCU or CCC or CCA or CCG

Threonine (Thr or T) ACU or ACC or ACA or ACG

Alanine (Ala or A) GCU or GCG or GCA or GCG

Tyrosine (Tyr or Y) UAU or UAC

Histidine (His or H) CAU or CAC

Glutamine (Gln or Q) CAA or CAG

Asparagine (Asn or N) AAU or AAC

Lysine (Lys or K) AAA or AAG

Aspartic acid (Asp or D) GAU or GAC

Glutamic acid (Glu or E) GAA or GAG

Cysteine (Cys or C) UGU or UGC

Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG

Glycine (Gly or G) GGU or GGC or GGA or GGG

Tryptophan (Trp or W) UGG

Stop codon UAA (ocher codon) or UAG (amber codon) or (milk white codon)

It should be understood that the above codons are for RNA sequences. The corresponding codon for DNA is to replace U with T.

In addition to the human GAVE18 nucleotide sequence shown in SEQ ID NO: 1, those skilled in the art can understand that there are DNA sequence polymorphisms that lead to changes in the amino acid sequence of GAyE18 in a population (such as a human population). Such genetic polymorphisms of the GAVE18 gene exist among individuals in a population due to natural allelic variation. An allele is one of a group of genes that can optionally be present at a particular locus. The terms "gene" and "recombinant gene" as used herein refer to a nucleic acid molecule comprising an open reading frame encoding a GAVE18 protein, preferably a mammalian GAVE18 protein. The phrase "allelic variant" as used herein refers to a nucleotide sequence located at the GAVE18 locus or to a polypeptide encoded by the nucleotide sequence. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be easily performed by using hybridization probes to identify the same loci in multiple individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations in GAVE18 that are derived from natural allelic variations and do not alter the functional activity of GAVE18 are included within the scope of the present invention.

Furthermore, variants of the isolated nucleic acid molecules of the invention are readily prepared by those skilled in the art using routine experimental techniques, such as site-directed mutagenesis.

antisense nucleotide sequence

The invention also extends to antisense nucleic acid molecules, ie, molecules that are complementary to an intended nucleic acid encoding a protein, such as to the coding strand of a double-stranded cDNA molecule or to an mRNA sequence. Antisense nucleic acids can thus hydrogen bond to the intended nucleic acid. The antisense nucleic acid may be complementary to the full-length GAVE18 coding strand or only a portion thereof, such as all or part of the protein coding region (or open reading frame). The antisense nucleic acid molecule can be antisense to the non-coding region of the coding strand where the nucleotide sequence encoding GAVE18 is located. Noncoding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences flanking the coding region that are not translated into amino acids.

Given the coding strand sequence (for example, SEQ ID NO: 1) disclosed herein encoding GAVE18, the antisense nucleic acid of the present invention can be designed according to the Watson and Crick base pairing principles. The antisense nucleic acid molecule can be complementary to the full-length coding region of GAVE18 mRNA, but is more preferably an oligonucleotide that is only antisense to a part of the coding region or non-coding region of GAVE18 mRNA. For example, antisense oligonucleotides can be complementary to the region near the translation start site of GAVE18 mRNA. Antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. Antisense nucleic acids of the present invention can be produced by chemical synthesis and enzymatic ligation reactions by procedures well known in the art. For example, antisense nucleic acids (such as antisense oligonucleotides) can be chemically synthesized from naturally occurring nucleotides or various modified nucleotides designed to increase the biological Stabilizing or increasing the physical stability of the duplex formed between the antisense and intended nucleic acid, such as phosphorothioate derivatives, phosphate derivatives and acridine substituted nucleotides can be used.

Examples of modified nucleotides that can be used to generate antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- -(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl queosine, Inosine, N ⁶ -isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine Purine, 3-methylcytosine, 5-methylcytosine, N ⁶ -adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2- Thiouracil, β-D-mannosyl queosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N ⁶ -prenyl adenine, uracil- 5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil , Uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine. Alternatively, antisense nucleic acids can be biologically produced using expression vectors in which the nucleic acid is subcloned in the antisense orientation (ie, the RNA transcribed from the inserted nucleic acid is in the antisense orientation to the target nucleic acid of interest).

The antisense nucleic acid molecules of the present invention are generally administered to a subject or produced in situ so as to hybridize or bind to cellular mRNA and/or genomic DNA encoding the GAVE18 protein, thereby inhibiting the expression of the protein by, for example, inhibiting transcription and/or translation. Express. Stable duplexes can be hybridized by conventional nucleotide complementarity, or by specific interactions in the major groove of the double helix, such as when an antisense nucleic acid molecule is used to bind the DNA duplex, or by hybridization to the regulatory region of GAVE18.

An example of a mode of administration of an antisense nucleic acid molecule of the invention includes direct injection at a tissue site. Additionally, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, an antisense molecule can be modified such that the molecule specifically binds to a receptor or antigen expressed on the surface of a selected cell, such as by linking the antisense nucleic acid molecule to a receptor that binds to a cell surface receptor. On the peptide or antibody of the body or antigen. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. In order to achieve a sufficient intracellular concentration of the antisense molecule, the antisense nucleic acid molecule can be placed under the control of a strong promoter, preferably a pol II or pol III promoter.

The antisense nucleic acid molecule of the present invention can be an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms special double-stranded hybrids with complementary RNA in which the strands run parallel to each other (Gaultier et al., Nucleic Acids Res (1987) 15:6625-6641). Antisense nucleic acid molecules may also comprise methyl ribonucleotides (Inoue et al., Nucleic Acids Research (1987) 15:6131-6148) or chimeric RNA-DNA analogs (Inoue et al., FEBS Lett (1987) 215: 327-330).

ribozyme

The present invention also includes ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that cleave single-stranded nucleic acids, such as mRNA, to which the ribozyme hybridizes. Thus, ribozymes such as the hammerhead ribozyme (Haselhoff et al., described in Nature (1988) 334:585-591 ) can be used to catalytically cleave the GAVE18 mRNA transcript, thereby inhibiting the expression of GAVE18 mRNA. translate. Ribozymes specific for nucleic acids encoding GAVE18 can be designed based on the nucleotide sequence of GAVE18 DNA disclosed herein (e.g., SEQ ID NO: 1). For example, derivatives of Tetrahymena L-19 IVS RNA can be constructed, wherein the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cut in the mRNA encoding GAVE18, see, for example, U.S. Patent Nos. 4,987,071 and 5,116,742, or, Catalytic RNAs with specific ribonuclease activity can be selected from a population of RNA molecules using GAVE18 mRNA, see, e.g., Bartel et al., Science (1993) 261:1411-1418.

Triple helix nucleic acid molecule and peptide nucleic acid of the present invention

The invention also includes nucleic acid molecules capable of forming triple helix structures. For example, gene expression of GAVE18 can be inhibited by orienting the nucleotide sequence complementary to the regulatory region of GAVE18 (such as the GAVE18 promoter and/or enhancer) to form a triple helix structure to prevent the expression of the GAVE18 gene in target cells. Transcription, see generally Helene, Anticancer Drug Des (1991) 6(6): 569; Helene Ann NY Acad Sci (1992) 660: 27; and Maher, Bioassays (1992) 14 (12):807.

In preferred embodiments, the nucleic acid molecules of the present invention may be modified at the base moiety, sugar moiety or phosphate backbone to improve, for example, the stability, hybridizability or solubility of the molecule. For example, the phosphate deoxyribose backbone of nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., Bioorganic & Medicinal Chemistry (1996) 4:5). The term "peptide nucleic acid" or "PNAs" as used herein refers to nucleic acid mimics, such as DNA mimics, in which the deoxyribose phosphate backbone is replaced with a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral PNA backbone has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. PNA can be performed by standard solid phase peptide synthesis as described by Hyrup et al. (1996) supra; Perry-O'Keefe et al., Proc Natl Acad Sci USA (1996) 93:14670 Synthesis of oligomers.

PNAs of GAVE18 can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific regulation of gene expression by, for example, inducing transcriptional or translational arrest or inhibiting replication. The PNA of GAVE18 can also be used. For example, PNA can be used to analyze single base pair mutations in genes by, for example, PNA-directed PCR clamping; PNA can be used as an artificial restriction enzyme when used in combination with other enzymes such as S1 nuclease (Hyrup et al. (1996) supra); or PNA can be used as a probe or primer for DNA sequencing and hybridization (Hyrup et al., (1996) supra; Perry-O'Keefe et al., (1996) supra).

In another embodiment, the PNA of GAVE18 can be modified by attaching lipophilic or other auxiliary groups to the PNA, by forming PNA-DNA chimeras, or by using liposomes or other drug delivery techniques known in the art To eg enhance stability, specificity or cellular uptake. See supra for Hyrup et al. (1996); Finn et al., Nucleic Acids Res. (1996) 24(17):3357-63; Mag et al., Nucleic Acids Res. (1989) 17:5973; and Peterser et al., Bioorganic MedChem Synthesis of PNA-DNA chimeras was performed as described by Lett (1975) 5:1119.

GAVE18 protein

Further, the present invention extends to an isolated polypeptide comprising the amino acid sequence of Figure 6 (SEQ ID NO: 2), variants, fragments, analogs or derivatives thereof.

The isolated nucleic acid molecule encoding the GAVE18 protein (such as a variant) having a sequence different from that of SEQ ID NO: 2 can be obtained by introducing one or more nucleotide substitutions, additions in the nucleotide sequence of SEQ ID NO: 1 or deletion, resulting in the introduction of one or more amino acid substitutions, additions or deletions in the encoded protein.

In a specific embodiment, the mutated GAVE18 protein can be tested for: (1) ability to form protein:protein interactions with proteins in the GAVE18 signaling pathway; (2) ability to bind GAVE18 ligand; or (3 ) ability to bind intracellular target proteins. In another embodiment, mutated GAVE18 proteins can be tested for their ability to regulate cell proliferation or cell differentiation.

Native GAVE18 protein can be isolated from cell or tissue sources by an appropriate purification strategy using standard protein purification techniques. Alternatively, GAVE18 proteins are readily produced by recombinant DNA techniques. As another alternative included in the present invention, the GAVE18 protein or polypeptide can be chemically synthesized using standard peptide synthesis techniques.

"Isolated" or "purified" protein, or biologically active portion thereof, is substantially free of cellular material or other contaminating proteins in the cell or tissue source from which the GAVE18 protein was derived, or when produced by chemical synthesis, substantially free of chemical precursors or other chemicals. The phrase "substantially free of cellular material" includes preparations of GAVE18 protein in which the protein is separated from the cellular components of the cells from which the protein was isolated or recombinantly produced. Accordingly, a GAVE18 protein that is substantially free of cellular material includes preparations of a GAVE18 protein that have less than about 30%, 20%, 10% or 5% or less (by dry weight) of non-GAVE18 proteins (also herein called "contaminating proteins"). When the GAVE18 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of medium, i.e., the medium occupies less than about 20%, 10%, 5% or less of the volume of the protein preparation . When the GAVE18 protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, ie, the protein is separated from the chemical precursors or other chemicals involved in its synthesis. Accordingly, such GAVE18 protein preparations have less than about 30%, 20%, 10% or 5% or less (by dry weight) of chemical precursors or non-GAVE18 chemicals.

Biologically active portions or fragments of the GAVE18 protein include peptides comprising an amino acid sequence sufficiently identical to or derived from the amino acid sequence of the GAVE18 protein (such as the amino acid sequence shown in SEQ ID NO: 2), and The peptide comprises fewer amino acids than the full-length GAVE18 protein and exhibits at least one activity of the GAVE18 protein. Generally, the biologically active portion comprises a domain or motif having at least one activity of the GAVE18 protein. The biologically active portion of the GAVE18 protein can be, for example, a polypeptide of 10, 25, 50, 100 or more amino acids in length. Preferred biologically active polypeptides include one or more of the identified GAVE18 domains.

Furthermore, other biologically active portions (in which other regions of the GAVE18 protein have been deleted) can be prepared by recombinant techniques and assessed against one or more functional activities of the native GAVE18 protein.

Other useful GAVE18 proteins are substantially the same as SEQ ID NO: 2 and retain the functional activity of the SEQ ID NO: 2 protein, but due to natural allelic variation or mutagenesis, its amino acid sequence differs from SEQ ID NO: 2. For example, such GAVE18 proteins and polypeptides have at least one biological activity described herein.

Accordingly, a useful GAVE18 protein is a protein comprising at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, 99% or 100% of the amino acid sequence of SEQ ID NO: 2 identical amino acid sequence, and retain the functional activity of the GAVE18 protein of SEQ ID NO:2. In a specific embodiment, the GAVE18 protein retains the functional activity of the GAVE18 protein of SEQ ID NO:2.

In order to determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned to optimize the comparison (for example, a gap (gap) can be introduced into the first amino acid or nucleic acid sequence to optimize the alignment with the second amino acid or nucleic acid sequence. sequence comparison of nucleic acid sequences). The amino acid residues or nucleotides are then compared at corresponding amino acid positions or nucleotide positions. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are considered to be identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences (ie, percent identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A specific non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm provided by Karlin et al., Proc. 1993) 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J Mol Bio (1990) 215:403. NBLAST program can be used to perform BLAST nucleotide search, setting such as score = 100, word length = 12, to obtain nucleotide sequences homologous to the GAVE18 nucleic acid molecule of the present invention. XBLAST program can be used to perform BLAST protein search, set score=50, word length=3, to obtain the amino acid sequence homologous to the GAVE18 protein molecule of the present invention. To obtain gapped aligned sequences for comparison, GappedBLAST as described in Altschul et al., Nucleic Acids Res. (1997) 25:3389 can be used. Alternatively, PSI-Blast can be used to perform an iterative search to detect distant relationships between molecules. Altschul et al. (1997) supra. When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used, see http://www.ncbi.nlm.nih.gov .

Another specific non-limiting example of a mathematical algorithm for comparing sequences is the algorithm provided by Myers et al., CABIOS (1988) 4: 11-17. This algorithm was introduced as part of the ALIGN program (version 2.0) of the GCG sequence comparison package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps. When calculating percent identity, only exact matches are counted.

The invention further extends to GAVE18 chimeras or fusion proteins. As used herein, a GAVE18 "chimeric protein" or "fusion protein" includes a GAVE18 polypeptide operably linked to a non-GAVE18 polypeptide. "GAVE18 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to GAVE18. A "non-GAVE18 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is substantially different from the GAVE18 protein, such as a protein that is different from the GAVE18 protein and is from the same or a different organism. In the GAVE18 fusion protein, the GAVE18 polypeptide may correspond to all or part of the GAVE18 protein, preferably at least one biologically active part of the GAVE18 protein. In fusion proteins, the term "operably linked" means that the GAVE18 polypeptide and the non-GAVE18 polypeptide are fused in frame with each other. A non-GAVE18 polypeptide can be fused to the N- or C-terminus of the GAVE18 polypeptide. One useful fusion protein is GST-GAVE18, wherein the GAVE18 sequence is fused to the C-terminus of glutathione-S-transferase (GST). This fusion protein allows easy purification of recombinant GAVE18.

In another embodiment, the fusion protein of the invention extends to a GAVE18-immunoglobulin fusion protein, wherein all or a portion of GAVE18 is fused to a sequence from a member of the immunoglobulin family. The GAVE18-immunoglobulin fusion protein of the present invention can be incorporated into a pharmaceutical composition and administered to a subject to inhibit the interaction between the GAVE18 ligand and the GAVE18 protein on the cell surface, thereby inhibiting GAVE18-mediated signal transduction in vivo guide. GAVE18-immunoglobulin fusion proteins can be used to affect the bioavailability of GAVE18-related ligands. Inhibition of GAVE18 ligand-GAVE18 interactions is useful therapeutically, for treating proliferation and differentiation disorders and for modulating (eg, promoting or inhibiting) cell survival. Furthermore, the GAVE18-immunoglobulin fusion proteins of the invention can be used as immunogens to generate anti-GAVE18 antibodies in a subject, to purify GAVE18 ligands, and to perform screening tests to determine compounds that inhibit the interaction between GAVE18 and GAVE18 ligands. molecular.

In a specific embodiment, the GAVE18 chimeric or fusion proteins of the invention are produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are ligated in reading frame according to conventional techniques, such as ligation with blunt or sticky ends, restriction enzyme digestion to provide suitable ends, blunt sticky ends as needed, alkaline phosphatase treatment To avoid unwanted ligation and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automatic DNA synthesizers. Alternatively, anchor primers can be used for PCR amplification of gene segments, which create complementary overhangs between two contiguous gene segments, which can then be annealed and reamplified to generate chimeric gene sequences (see e.g. Ausubel et al., see above). Furthermore, a number of expression vectors are commercially available that encode fusion moieties such as GST polypeptides. Nucleic acid encoding GAVE18 can be cloned into such expression vectors such that the fusion moiety is linked in frame with the GAVE18 protein.

Variants

As mentioned above, the present invention further extends to GAVE18 protein variants. For example, mutations can be introduced in the amino acid sequence of SEQ ID NO: 2 by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, one or more amino acids may be replaced by another amino acid of similar polarity as a functional equivalent, resulting in a silent change. The amino acid to be substituted in the amino acid sequence of the polypeptide of the present invention may be selected from other members of the group to which the amino acid belongs. Examples of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing an aromatic ring structure are phenylalanine, tryptophan and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes are not expected to affect apparent molecular weight or isoelectric point as determined by polyacrylamide gel electrophoresis.

Particularly preferred replacements are:

Replacing Arg with Lys, or vice versa, still maintains a positive charge;

Replace Asp with Glu, or vice versa, so that the negative charge is still maintained;

Replace Thr with Ser so that free OH is still maintained; and

Asn is replaced with Gln so that free _NH2 is still maintained.

Furthermore, amino acid substitutions may be introduced to replace amino acids with particularly preferred properties. For example, a Cys can be introduced into a potential site for forming a disulfide bridge with another Cys. His can be introduced as a specific "enzymatic" site (ie His can act as an acid or a base and is the most common amino acid in biochemically catalyzed reactions). Pro can be imported because of its specific planar structure that induces β-turns in protein structures.

Mutations can also be randomly introduced into the full-length or part of the GAVE18 coding sequence, such as by saturation mutagenesis, and mutants that retain activity can be identified by screening the resulting mutants for GAVE18 biological activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

The variants of the invention may function as GAVE18 agonists (mimetics) or as GAVE18 antagonists. Variants of the GAVE18 protein can be produced by mutagenesis, such as discrete point mutations or truncations of the GAVE18 protein. An agonist of the GAVE18 protein can retain substantially the same biological activity or a portion of the biological activity of the naturally occurring GAVE18 protein. Antagonists of the GAVE18 protein can inhibit one or more activities of the naturally occurring form of the GAVE18 protein by, for example, competitively binding to downstream or upstream members of a cellular signaling cascade involving the GAVE18 protein. Thus, treatment with functionally restricted variants can produce specific biological effects. Treatment of a subject with a variant that has a fraction of the biological activity of the naturally occurring form of the GAVE18 protein may result in fewer adverse effects on the subject than treatment with the naturally occurring form of the protein.

GAVE18 protein variants that function as GAVE18 agonists (mimetics) or GAVE18 antagonists can be identified by screening combinatorial libraries of GAVE18 protein mutants, such as truncation mutants, for GAVE18 agonist or antagonist activity. In one embodiment, a diverse library of GAVE18 variants is generated by combinatorial mutagenesis at the nucleic acid level and encoded by a diverse library of genes. For example, a diverse library of GAVE18 variants can be prepared by enzymatically ligating a mixture of synthetic oligonucleotides into a gene sequence such that a degenerate set of potential GAVE18 sequences can be expressed as a single polypeptide, or alternatively Alternatively, expressed as a set of larger fusion proteins (eg, phage display) that contain the set of GAVE18 sequences therein. There are a number of methods that can be used to generate a library of potential GAVE18 variants from degenerate oligonucleotide sequences. The chemical synthesis of the degenerate gene sequence can be carried out on an automatic DNA synthesizer, and then the synthesized gene can be ligated into a suitable expression vector. The use of a degenerate set of genes makes it possible to provide in one mixture all sequences encoding the desired set of potential GAVE18 sequences. Methods for the synthesis of degenerate oligonucleotides are well known in the art (see e.g. Narang, Tetrahedron (1983) 39:3; Itakura et al, Ann Rev Biochem (1984) 53:323; Itakura et al., Science (1984) 198:1056; Ike et al., Nucleic Acids Res. (1983) 11:477).

In addition, libraries of fragments of the GAVE18 protein coding sequence can be used to generate a diverse population of GAVE18 fragments for screening and subsequent selection of GAVE18 protein variants. In one embodiment, a library of fragments of coding sequences can be generated by nuclease-treating double-stranded PCR fragments of the GAVE18 coding sequence (under conditions in which only about one cleavage occurs per molecule), denaturing The double-stranded DNA, the resulting DNA is annealed to form a double-stranded DNA that may contain sense/antisense pairs from different nick products, and the re-formed duplex is treated with S1 nuclease to remove the single-stranded portion and the resulting fragment library ligated into the expression vector. By this method, an expression library encoding the N-terminal and internal fragments of different sizes of the GAVE18 protein can be obtained.

Several techniques are known in the art for screening gene products of combinatorial libraries prepared by point mutations or truncations and for screening cDNA libraries for gene products with selected properties. Such techniques are suitable for rapid screening of gene libraries generated by combinatorial mutagenesis of GAVE18 proteins. The most widely used technique suitable for high-throughput analysis and screening of large gene libraries generally involves cloning the gene library into a replicable expression vector, transforming suitable cells with the resulting vector library, and expressing the combined gene under certain conditions, wherein all Detection of desired activity facilitates the isolation of vectors encoding genes whose expression products have been detected. Recursive ensemble mutagenesis (REM) is a technique for increasing the frequency of functional mutants in libraries that can be used in conjunction with screening tests to identify GAVE18 variants (Arkin et al., PNAS USA (1992) 89 : 7811-7815; Delgrave et al., Protein Engineering (Protein Engineering) (1993) 6(3):327-331).

GAVE18 ANALOGS AND DERIVATIVES

Furthermore, the present invention also includes GAVE18 derivatives or analogs produced by chemical modification. The GAVE18 protein can be derived by linking one or more molecules to the molecule.

Chemical molecules for derivatization: Chemical molecules suitable for derivatization may be selected from water-soluble polymers such that the GAVE18 analogue or derivative does not precipitate in an aqueous environment such as a physiological environment. Optionally, the polymer is pharmaceutically acceptable. Those skilled in the art can base their considerations on whether the polymer/component linker is therapeutically useful, and if so, target dosage, circulation time, resistance to proteolysis, etc. For GAVE18, this can be determined by applying the assay presented here. Examples of water-soluble polymers that have been used herein include, but are not limited to, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 , 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), dextran, poly(n- vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol or polyvinyl alcohol. Polyethylene glycol propionaldehyde has manufacturing advantages due to its stability in water.

The polymers can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 2 kDa and about 100 kDa (the term "about" means that at the time the polyethylene glycol was made, some molecules had a molecular weight greater than that indicated and some molecules had a molecular weight less than that indicated) , due to ease of handling and fabrication. Other molecular weight sizes may be used, depending on the desired therapeutic properties (e.g., duration of desired sustained release, effect on biological activity (if any), ease of handling, degree or lack of antigenicity, and polyethylene other known effects of diols on therapeutic proteins or analogs).

The number of polymer molecules attached to GAVE18 is variable and the effect on function can be determined by one skilled in the art. GAVE18 may be mono-derivatized with the same or different chemical molecules (eg, polymers, such as polymers with different polyethylene glycol molecular weights), or may provide di-, tri-, tetra- or some combination of derivatizations. The ratio of polymer molecules to GAVE18 molecules can vary, which ratio will be their concentration in the reaction mixture. Typically, the optimum ratio (no excess of unreacted components or components and polymer, depending on reaction efficiency) will be determined by factors such as the degree of derivatization targeted (e.g., mono-, di-, tri-derivatization, etc.), the desired The molecular weight of the polymer chosen, whether the polymer is branched or unbranched, and the reaction conditions.

The connection of polyethylene glycol molecules (or other chemical molecules) to GAVE18 should consider its influence on the functional domain or antigenic domain of GAVE18. A number of linking methods are available to those skilled in the art, such as EP 0401 384 (herein incorporated by reference) (coupling PEG to G-CSF), see also Malik et al., 1992, Exp.Hematol.20: 1028-1035 ( reported PEGylation of GM-CSF with tresyl chloride). For example, polyethylene glycol can be covalently attached via reactive groups of amino acid residues such as free amino or carboxyl groups. A reactive group is a group that can attach an activated polyethylene glycol molecule. Amino acid residues with free amino groups include lysine residues and N-terminal amino acid residues; amino acid residues with free carboxyl groups include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues. Sulfhydryl groups can also be used as reactive groups for attachment of polyethylene glycol molecules. For therapeutic purposes, attachment at an amino group, such as at the N-terminus or a lysine group, is preferred.

Particularly desirable is N-terminal chemically modified GAVE18. Polyethylene glycol is used to describe the composition, and various polyethylene glycol molecules can be selected (according to their molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to GAVE18 molecules in the reaction mixture, the PEG to be carried out type of reaction, and methods to obtain selected N-terminal PEGylated proteins. A method of obtaining N-terminally PEGylated preparations (ie, separating N-terminally PEGylated molecules from other mono-PEGylated molecules if desired) may be by purifying N-terminally PEGylated species from a population of PEGylated protein molecules. Selective N-terminal chemical modification can be performed by reductive alkylation, which takes advantage of the difference in reactivity of the different types of primary amino groups (lysine vs. N-terminal) that can be used to derivatize GAVE18. Under suitable reaction conditions, GAVE18 can be derivatized substantially selectively at the N-terminus of GAVE18 with a carboxyl-containing polymer. For example, the selective N-terminally PEGylated GAVE18 can be reacted at a pH that allows the use of the difference between the pKa of the GAVE18 lysine residue ε-amino group and the pKa of the GAVE18 N-terminal residue α-amino group. . Through such selective derivatization, the connection of water-soluble polymers to GAVE18 can be controlled: the binding to the polymer mainly occurs at the N-terminus of GAVE18, and other reactive groups such as the side chain amino groups of lysine are not significantly modified . When reductive alkylation is used, the water soluble polymer can be of the type described above and have a single reactive aldehyde for coupling to GAVE18. Polyethylene glycol propionaldehyde, which contains a single reactive aldehyde, can be used.

GAVE18 antibodies, variants, fragments, analogs or derivatives thereof

Isolated GAVE18 proteins or portions or fragments thereof can be used as immunogens to generate antibodies that bind GAVE18 using standard polyclonal and monoclonal antibody preparation techniques. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that specifically binds an antigen such as GAVE18 or a fragment thereof. A molecule that specifically binds GAVE18 is a molecule that binds GAVE18 without substantially binding other molecules in a sample that naturally contains GAVE18, such as a biological sample. Examples of immunologically active portions of immunoglobulin molecules include F _(ab) and F _(ab')2 fragments which can be produced by treatment of antibodies with enzymes such as pepsin. The present invention provides polyclonal antibodies, monoclonal antibodies and chimeric antibodies using GAVE18 antibody, its variants, fragments, analogs or derivatives as immunogens. Chimeric antibodies are preferred for use in the treatment of human diseases or disorders because human or humanized antibodies are far less likely by themselves to induce an immune response, especially an allergic response, than xenogeneic antibodies.

The full length GAVE18 protein can be used or, alternatively, the invention provides antigenic peptide fragments of GAVE18 for use as immunogens. The antigenic peptide of GAVE18 comprises at least 8 (preferably 10, 15, 20, 30 or more) amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and contains a GAVE18 epitope, such that the resulting Antibodies against this peptide can form specific immune complexes with GAVE18.

The GAVE18 immunogen is generally used to prepare antibodies by immunizing a suitable subject (eg, rabbit, goat, mouse or other mammal) with the immunogen. Suitable immunogenic preparations may comprise, for example, recombinantly expressed GAVE18 protein or chemically synthesized GAVE18 polypeptide. The preparation may also contain an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant. Immunization of a suitable subject with an immunogenic GAVE18 preparation induces a polyclonal anti-GAVE18 antibody response.

Antibodies of the invention may be monoclonal antibodies, polyclonal antibodies or chimeric antibodies. The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a population of antibody molecules that contain only one antigen binding site that is immunoreactive with a specific epitope of GAVE18. Such monoclonal antibody compositions typically exhibit a single binding affinity for a particular GAVE18 protein epitope.

Polyclonal anti-GAVE18 antibodies can be prepared as described above by immunizing a suitable subject with the GAVE18 immunogen. Antibody titers to GAVE18 can be monitored over time in immunized subjects using standard techniques, such as an enzyme-linked immunosorbent assay (ELISA) using immobilized GAVE18. If desired, antibody molecules directed against GAVE18 can be isolated from mammals (eg, from blood) and further purified using well-known techniques, such as protein A chromatography, to obtain an IgG fraction. At an appropriate time after immunization, such as when the anti-GAVE18 antibody titer is highest, antibody-producing cells can be obtained from the subject and used to produce monoclonal antibodies by standard techniques such as Kohler et al., Nature (Nature ) (1975) 256:495-497 originally described hybridoma technology, human B cell hybridoma technology (Kohler et al., Immunology Today (ImmunolToday) (1983) 4:72), EBV hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy), (1985), Alan R. Liss, Inc., pp. 77-96) or trioma technology. Techniques for generating hybridomas are well known (see generally: Current Protocols in Immunology (1994) Coligan et al., eds., John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (usually myeloma) was fused with lymphocytes (usually splenocytes) from a mammal immunized with the GAVE18 immunogen as described above, and the resulting hybridoma culture supernatants were screened to identify hybridomas producing monoclonal antibodies that bind GAVE18.

Anti-GAVE18 monoclonal antibodies can be generated using any of the well-known methods for fusing lymphocytes and immortal cell lines (see e.g. Current Techniques in Immunology, supra; Galfre et al., Nature (1977) 266:550-552; Kenneth , Monoclonal Antibodies: A New Dimension In Biological Analyzes, Plenum Publishing Corp., New York, N.Y (1980); and Lerner, Yale J Biol Med ( 1981) 54:387-402). Moreover, those of ordinary skill will appreciate that many variations of such methods may also be used. Typically immortal cell lines (eg, myeloma cell lines) are derived from the same mammalian species as lymphocytes. For example, murine hybridomas can be prepared by fusing lymphocytes of mice immunized with the immunogenic preparation of the invention with an immortal mouse cell line, such as a myeloma cell line, wherein the myeloma cell line contains a pair of hypoxanthine, aminopterin, and thymidine-based medium ("HAT medium"). Any of a number of myeloma cell lines can be used as fusion partners according to standard techniques, such as P3-NS1/l-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma cells Tie. These myeloma cell lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells and mouse splenocytes are fused with polyethylene glycol ("PEG"). The resulting hybridoma cells from the fusion are then selected with HAT medium, which kills unfused and non-productively fused myeloma cells (unfused splenocytes die after a few days due to not being transformed). Monoclonal antibody-producing hybridoma cells of the invention can be detected by screening myeloma cell culture supernatants for antibodies that bind to GAVE18 (eg, using a standard ELISA assay).

As an alternative to producing monoclonal antibody-secreting hybridomas, GAVE18 can be used to screen recombinant combinatorial immunoglobulin libraries (such as antibody phage display libraries), thereby allowing the isolation of immunoglobulin library members that bind GAVE18, thereby identifying and isolating Anti-GAVE18 monoclonal antibody. Kits for generating and screening phage display libraries are available from commercial suppliers (eg, Pharmacia Recombinant Phage Antibody System, Cat. No. 27-9400-01; and Stratagene "SURFZAP" Phage Display Kit, Cat. No. 240612).

In addition, examples of methods and reagents particularly suitable for generating and screening antibody display libraries can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/17271; 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; (1991) 9: 1370-1372; Hay et al., Hum Antibody Hybridomas (1992) 3: 81-85; Huse et al., Science (1989) 246: 1275-1281 and Griffiths et al., EMBO J (1993) 25( 12): 725-734.

In addition, recombinant anti-GAVE18 antibodies, such as chimeric and humanized monoclonal antibodies (comprising both human and non-human portions) can be prepared using standard recombinant DNA techniques. The chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using the methods described in: PCT Publication No. WO 87/02671; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application No. 125,023; Better et al., Science (1988) 240:1041-1043; ) 84:3439-3443; Lin et al., J. Immunology (1987) 139:3521-3526; Sun et al., Proceedings of the National Academy of Sciences USA (1987) 84:214-218; Nishimura et al., Cancer Research (Cane Res) (1987 )47:999-1005; Wood et al., Nature (1985) 314:446-449; Shaw et al., J Natl Cancer Inst (1988) 80:1553-1559; Morrison, Science (1985) 229 : 1202-1207; Oi et al., Bio/Techniques (1986) 4: 214; US Pat. No. 5,225,539; Jones et al., Nature (1986) 321: 552-525; Verhoeyan et al., Science (1988) 239: 1534; and Beidler et al., J Immunol (1988) 141:4053-4060.

There is a particular need for fully human antibodies for use in the treatment of human patients. Such antibodies can be prepared using transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes but are capable of expressing human heavy and light chain genes. The transgenic mice can be immunized with the antigen of choice (eg, all or a portion of GAVE18) in a standard manner. Monoclonal antibodies directed against this antigen can be obtained by conventional hybridoma technology. Human immunoglobulin transgenes located in transgenic mice undergo rearrangement during B-cell differentiation and subsequently undergo class switching and somatic mutation. Thus, use of this epitope allows the identification of antibodies that inhibit GAVE18 activity. The heavy and light chains of non-human antibodies were cloned and used to construct phage-displayed F _ab fragments. For example, the heavy chain gene can be cloned into a plasmid vector such that the bacteria secrete the heavy chain. The light chain gene can be cloned into the phage coat protein gene so that it can be expressed on the phage surface. Bacteria expressing non-human heavy chains were infected with phage fused to a library of human light chains (collected at random). The resulting progeny phage display hybrid antibodies (human light chain/non-human heavy chain). The selected antigen is used to pan for phage that bind the selected antigen. Several rounds of selection may be required to identify the phage.

The human light chain gene is isolated from phage selected to bind the selected antigen. The selected human light chain genes were then used to guide the selection of human heavy chain genes, as described below. The selected human light chain genes were inserted into bacterial expression vectors. Bacteria expressing selected human light chains are infected with phage fused to the human heavy chain library. The resulting progeny phage display human antibodies (human light chain/human heavy chain).

Next, the selected antigens are used to pan for phage that can bind to the selected antigen. The selected phage display fully human antibodies that recognize the same epitope as the originally selected non-human monoclonal antibody. The genes encoding the heavy and light chains are isolated and can be further manipulated for the production of human antibodies. This technique is described by Jespers et al. (Bio/Technology (1994) 12:899-903).

Anti-GAVE18 antibodies (eg, monoclonal antibodies) can be used to isolate GAVE18 by standard techniques (eg, affinity chromatography or immunoprecipitation). Anti-GAVE18 antibodies can help purify native GAVE18 from cells and recombinantly produced GAVE18 expressed in host cells. In addition, anti-GAVE18 antibodies can be used to detect GAVE18 protein (eg, in cell lysates or cell supernatants) to assess the abundance and expression pattern of GAVE18 protein. Anti-GAVE18 antibodies are useful in diagnostics as part of a clinical testing procedure to monitor protein levels in tissue, for example, to determine the efficacy of a particular treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance, as described below.

detectable marker

Optionally, isolated nucleic acid molecules of the invention, polypeptides of the invention, antibodies of the invention, and fragments thereof may be detectably labeled. Suitable labels include enzymes, fluorophores such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), Texas Red (TR), rhodamine, free or chelated lanthanide salts (especially Eu ^{3 +} ), here are just some fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (such as biotin), bioluminescent materials and chemiluminescent agents. When a control label is used, the same or different labels can be used for the receptor and control label.

When using radioactive labels such as isotopes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re, available The counting methods that can be used are known. When the label used is an enzyme, detection can be accomplished by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gas analytical techniques known in the art.

Direct labels are one example of labels that can be used in the present invention. Direct markers have been defined as entities that, in their native state, are readily visible with the naked eye or with the aid of optical filters and/or applied stimuli such as excitation of fluorescence with ultraviolet light. Examples of color markers that can be used in the present invention include metal sol particles such as gold sol particles described by Leuvering (US Patent 4,313,734); Gribnau et al. (US Patent 4,373,932) and May et al. (WO 88/08534) Dye sole particles; dyed latex as described by May (see above) and Snyder (EP-A 0 280 559 and 0281 327); or packets as described by Campbell et al. (US Patent 4,703,017) Dye encapsulated in liposomes. Other direct labels include radioactive nucleotides, fluorescent or luminescent molecules. In addition to these direct labels, indirect labels comprising enzymes are also useful in the present invention. Various types of enzyme-linked immunoassays are known in the art, such as alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been described by Eva Engvall described in enzyme immunoassays ELISA and EMIT (Methods in Enzymology, 70, 419-439, 1980 and US Patent 4,857,453).

Other markers useful in the present invention include magnetic resonance imaging markers.

In another embodiment, phosphorylation sites can be formed on the isolated polypeptide of the present invention, the antibody of the present invention or fragments thereof for ³² P labeling, such as in Sidney Pestka's European Patent No. 0372707 (Application No. 89311108.8), Or as described in U.S. Patent 5,459,240 (issued October 17, 1995 to Foxwell et al.).

As exemplified herein, proteins (including antibodies) can be labeled with metabolic markers. Metabolic labeling can occur in vitro during incubation of cells expressing the protein in media supplemented with metabolic markers such as [ ³⁵ S]-methionine or [ ³² P]-orthphosphate. In addition to metabolic (or biosynthetic) labeling with [ ³⁵ S]-methionine, the present invention also contemplates the use of [ ¹⁴ C]-amino acids and [ ³ H]-amino acids (replaced with tritium at positions that are less likely to change) .

Recombinant Expression Vectors and Host Cells

Another aspect of the invention relates to a vector, preferably an expression vector, comprising a nucleic acid encoding GAVE18 (or a portion thereof). As noted above, one type of vector is a "plasmid," which is a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors replicate autonomously in host cells (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the host cell genome when introduced into the host cell and thus replicate along with the host genome. Furthermore, certain vectors (expression vectors) are capable of directing the expression of genes to which they are operably linked. In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which perform equivalent functions.

The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expression in a host cell. This means that the recombinant expression vector of the present invention includes one or more regulatory sequences selected based on the host cell used for expression, which sequence is operably linked to the nucleic acid to be expressed. In a recombinant expression vector, "operably linked" means that a nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or when the vector is introduced into a host expressed in the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in a variety of host cells (eg, tissue-specific regulatory sequences). Those skilled in the art will understand that the design of the expression vector will depend on factors such as the host cell selected for transformation, the expression level of the desired protein, and the like. The expression vectors of the present invention can be introduced into host cells to produce proteins or peptides encoded by the nucleic acids described herein (eg GAVE18 protein, mutant forms of GAVE18 and fusion proteins, etc.).

The recombinant expression vector of the present invention can be designed to express GAVE18 in prokaryotic or eukaryotic cells, such as bacterial cells (such as Escherichia coli), insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. cell. Suitable host cells are discussed previously in Goeddel. Alternatively, recombinant expression vectors can be transcribed and translated in vitro using regulatory elements and proteins such as bacteriophage, such as T7 promoter and/or T7 polymerase.

Expression of proteins in prokaryotic cells is mostly carried out in E. coli using vectors containing constitutive or inducible promoters directing the expression of fusion or non-fusion proteins. Fusion vectors add amino acids to the protein they encode, usually to the amino terminus of the recombinant protein. Such fusion vectors generally have three uses: 1) to increase the expression of recombinant proteins; 2) to increase the solubility of recombinant proteins; and 3) to assist in the purification of recombinant proteins as ligands in affinity purification. Usually, a proteolytic cleavage site is introduced at the junction of the fusion part and the recombinant expressed protein in the fusion expression vector, so that the recombinant protein can be separated from the fusion part after purification of the fusion protein. Such enzymes and associated recognition sequences include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith et al., Gene (1988) 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ), three The latter fused glutathione S-transferase (GST), maltose E-binding protein or protein A to the target recombinant protein, respectively.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Enzymatic Methods: Methods in Enzymology), Academic Press, San Diego, California (1990) 185:60-89). Expression of target genes from the pTrc vector relies on host RNA polymerase to initiate transcription from a hybrid trp-lac fusion promoter.

One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host whose ability to proteolytically cleave the recombinant protein is impaired (Gottesman, Gene Expression Technology: An Enzymatic Approach, Academic Press, San Diego, California (1990) 185 : 119-128). Another strategy is to change the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that each codon encoding each amino acid is a codon preferably used in E. coli (Wada et al., Nucleic Acids Research (1992) 20:2111-2118 ). Such alterations to the nucleic acid sequences of the invention can be accomplished by standard DNA synthesis techniques.

In another embodiment, the GAVE18 expression vector is a yeast expression vector. Examples of vectors for expression in yeast (such as S. cerevisiae) include pYepSecl (Baldari et al., EMBO J (1987) 6:229-234), pMFa (Kurjan et al., Cell (Cell) (1982 ) 30:933-943), pJRY88 (Schultz et al., Gene (1987) 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA) and pPicZ (Invitrogen Corp, San Diego, CA).

Alternatively, GAVE18 can be expressed in insect cells using a baculovirus expression vector. Existing baculovirus vectors that can be used to express proteins in cultured insect cells (such as Sf 9 cells) include the pAc series (Smith et al., Molecular Cell Biology (Mol Cell Biol) (1983) 3: 2156-2165) and pVL series (Lucklow et al., Virology (1989) 170:31-39).

In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840) and pMT2PC (Kaufman et al., EMBO J (1987) 6:187-195). When used in mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. previously.

In another embodiment, a recombinant mammalian expression vector is capable of directing the expression of a nucleic acid preferentially in a particular cell type (eg, using tissue-specific regulatory elements to express the nucleic acid). Tissue-specific regulatory elements are well known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., Genes Dev (1987) 1:268-277), the lymphoid-specific promoter (Calame et al., Adv Immunol (1988) 43:235-275), especially T cell receptors (Winoto et al., EMBO J (1989) 8:729-733) and immunoglobulins (Banerji et al., Cell (Cell) (1983) 33:729- 740; Queen et al., Cell (Cell) (1983) 33:741-748), neuron-specific promoters (such as the neurofilament promoter; Byrne et al., Proceedings of the National Academy of Sciences of the United States (Proe Natl Acad USA) ( 1989) 86:5473-5477), pancreas-specific promoters (Edlund et al., Science (Science) (1985) 230:912-916) and mammary gland-specific promoters (such as the milk whey protein promoter; U.S. Patent No. 4,873,316 and European Application No. 264,166). Developmentally regulated promoters are also included, such as the mouse hox promoter (Kessel et al., Science (1990) (1990) 249:374-379) and the alpha-fetoprotein promoter (Campes et al., Genes Dev) (1989 )3:537-546).

The present invention also provides a recombinant expression vector containing the DNA molecule of the present invention, wherein the DNA molecule is cloned in the expression vector in an antisense direction. That is, the DNA molecule is operably linked to regulatory sequences in such a way that expression (by transcription of the DNA molecule) produces an RNA molecule that is antisense to GAVE18 mRNA. Regulatory sequences operably linked to nucleic acids cloned in the antisense orientation can be selected to direct the sustained expression of the antisense RNA molecules in a variety of cell types. For example, viral promoters and/or enhancers or regulatory sequences can be selected to direct constitutive, tissue-specific or cell-type-specific expression of the antisense RNA. The antisense expression vector can be in the form of recombinant plasmid, phagemid or attenuated virus, wherein the antisense nucleic acid is placed under the control of the high-efficiency regulatory region, and its activity is determined by the type of cell into which the vector is introduced. For a discussion of regulation of gene expression using antisense genes, see Weintraub et al. (Reviews-Trends in Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that the term refers not only to the particular subject cell, but also to the progeny or potential progeny of that cell. Certain changes may occur during passage, due to mutations or environmental influences, such that the progeny may not in fact be identical to the parental cells, but are still encompassed within the scope of the term as used herein.

The host cell can be any prokaryotic or eukaryotic cell. For example, GAVE18 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO), 293 cells or COS cells). Other suitable host cells are known to those skilled in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various techniques known in the art for introducing exogenous nucleic acid (such as DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, transduction, DEAE - Dextran-mediated transfection, lipofection or electroporation.

For stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into the genome depending on the expression vector and transfection technique applied. To identify and screen integrants, generally a gene encoding a selectable marker (such as an antibiotic resistance gene) is introduced into the host cell together with the gene of interest. Preferred selectable markers include those genes that confer resistance to drugs such as G418, hygromycin and methotrexate. The nucleic acid encoding the selectable marker may be introduced into the host cell on the same vector as the gene encoding GAVE18 or may be introduced on a different vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells incorporating the selectable marker gene will survive while other cells die).

The host cells (eg, cultured prokaryotic or eukaryotic host cells) of the invention can be used to produce (ie, express) the GAVE18 protein. Therefore, the present invention further provides a method for producing GAVE18 protein by using the host cell of the present invention. In one embodiment, the method comprises culturing the host cell of the present invention into which a recombinant expression vector encoding GAVE18 has been introduced in a suitable medium, thereby allowing the GAVE18 protein to be produced. In another embodiment, the method further comprises isolating GAVE18 from the culture medium or host cells.

In another embodiment, GAVE18 is included in an inducible expression system for recombinant expression of other proteins subcloned into modified expression vectors. For example, host cells containing mutant G proteins (e.g., yeast cells, Y2 adrenocortical cells, and cyc ^- S49, see U.S. Pat. ) 89 (19): 8933-37 and Katada et al., Biochemical Journal (J BiolChem) (1984) 259 (6): 3586-95) transduce with the first expression vector containing the nucleic acid sequence of encoding GAVE18, wherein the GAVE18 is functionally expressed in host cells. Although expressed GAVE18 is constitutively active, the presence of the mutation does not allow signal transduction to occur; ie, the G protein-directed downstream cascade cannot be activated (eg, adenylyl cyclase cannot be activated). Subsequently, host cells containing GAVE18 are transduced with a second expression vector. In addition to the gene of interest to be expressed by this inducible system, the second vector contains a structural gene complementary to the host cell G protein mutant (i.e., a functional G _s , G _i , G _o or G of mammalian or yeast _q , see, eg, PCT Publication No. WO 97/48820; US Patent Nos. 6,168,927 Bl, 5,739,029, and 5,482,835). The complementary structural genes of the second carrier are inducible; that is, under the control of exogenously added compounds (such as tetracycline, IPTG, small molecules, etc., see the previous literature of Sambrook et al.), which can activate the A promoter to which a complementary structural gene is operably linked. Upon addition of an inducer, functional expression of the protein encoded by this complementary structural gene will result in constitutively active GAVE18 forming a complex leading to activation of the appropriate downstream pathway (eg, formation of a second messenger). The gene of interest contained in the second vector has an operably linked promoter that can be activated by a suitable second messenger (such as CREB and AP1 elements). Thus, when the second messenger accumulates, the promoter upstream of the gene of interest is activated to express the product of that gene. In the absence of an inducer, expression of the gene of interest is turned off.

In a preferred embodiment, the host cells used in this inducible expression system include, but are not limited to, S49(cyc ⁻ ) cells. Although cell lines containing G-protein mutations are contemplated by the present invention, suitable mutants can also be artificially prepared/constructed (see US Patent Nos. 6,168,927 Bl, 5,739,029, and 5,482,835 for yeast cells).

In a related aspect, cells are transformed with a vector operably linked to a cDNA comprising the sequence encoding the protein shown in SEQ ID NO:2. The first and second vectors included in the system can be considered to include, but are not limited to, pCDM8 (Seed, Nature (1987) 329:840) and pMT2PC (Kaufman et al., EMBO J (1987) 6:187-195), pYepSecl (Baldari et al., EMBO J (1987) 6:229-234), pMFa (Kurjan., Cell (1982) 30:933-943), pJRY88 (Schultz et al., Gene (1987) 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).

In a related aspect, host cells can be transfected by a suitable method, wherein the transfection results in the expression of a functional GAVE18 protein (e.g., Sambrook et al., supra and Kriegler, Gene Transfer and Expression: A Laboratory Guide (Gene Transfer and Expression : A Laboratory Manual), Stockton Press, New York, NY, 1990). The "functional protein" includes, but is not limited to, a protein that upon expression forms a complex with a G-protein, wherein the G-protein regulates the formation of a second messenger. Other methods of transfecting host cells that have been used here include, but are not limited to, transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, lipofection (lysosome fusion), the use of gene guns or DNA vector transporters (see e.g. Wu et al., 1992, J.Biol.Chem.267:963-967; Wu and Wu, 1988, J.Biol.Chem.263:14621-14624 ; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).

A variety of promoters are used in the present invention. Indeed, expression of the polypeptides of the invention can be regulated by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host chosen for expression. Promoters that can be used to regulate the expression of GAVE18 include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the Rous sarcoma virus 3' long terminal repeat (Yamamoto et al. , 1980, Cell 22:787-797), herpes thymidine kinase promoter (Wagner et al., 1981, Proc.Natl.Acad.Sci.U.S.A.78:1441-1445), the regulatory sequence of metallothionein gene (Brinster et al. People, 1982, Nature 296:39-42); prokaryotic expression vectors such as β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc.Natl.Acad.Sci.U.S.A.75:3727-3731) or tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 21-25); see also Scientific American, 1980, 42: 74-94, "Proteins of Use from Recombinant Bacteria"; From Yeast or Other Fungi Promoter elements such as Gal 4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and animal transcription that show tissue specificity and have been used in transgenic animals Regulatory region: Elastase I gene regulatory region active in pancreatic alveolar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 ; MacDonald, 1987, Hepatology 7:425-515); Insulin gene regulatory region active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin active in lymphoid cells Gene regulatory regions (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), in Mouse mammary cancer virus regulatory region active in testis, mammary gland, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene regulatory region active in liver (Pinkert et al., 1987, Genes and Devel.1: 268-276), active alpha-fetoprotein gene regulatory region in the liver (Krumlauf et al., 1985, Mol.Cell.Biol.5: 1639-1648; Hammer et al., 1987, Science 235:53-58), α1-antitrypsin gene regulatory region active in liver (Kelsey et al., 1987, Genes and Devel.1:161-171), β-antitrypsin active in myeloid cells Globulin gene regulatory region (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic protein active in oligodendrocytes of the brain Regulatory region (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314:283-286), and in the hypothalamus The active gonadotropin-releasing hormone gene regulatory region (Mason et al., 1986, Science 234:1372-1378).

Expression vectors comprising nucleic acid molecules of the present invention can be identified by four commonly used methods: (a) PCR amplification of target plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selectable marker gene function, and ( d) Expression of the inserted sequence. In the first method, nucleic acid can be amplified by PCR to provide an amplification product for detection. In the second method, the presence of the inserted foreign gene in the expression vector can be detected by nucleic acid hybridization using a probe comprising a sequence homologous to the inserted marker gene. In a third approach, recombinant vector/host systems can be identified and selected based on the presence or absence of certain "selectable marker" gene functions that are determined by the insertion of foreign genes into the vector. (eg, β-galactosidase activity, thymidine kinase activity, antibiotic resistance, transformed phenotype, inclusion body formation in baculovirus, etc.). In another example, if the nucleic acid encoding the GAVE18 protein, its variants, its analogs or its derivatives is inserted into the "selectable marker" gene sequence of the vector, the recombinant containing the insert can pass the GAVE18 gene function Identified by the lack of. In a fourth aspect, recombinant expression vectors can be identified by assaying the activity, biochemical or immunological properties of the gene product expressed by the recombinant if the expressed protein has a functionally active conformation.

A large number of host/expression vector combinations are available for expressing the DNA sequences of the invention. Useful expression vectors may consist, for example, of chromosomal segments, non-chromosomal segments and synthetic DNA sequences. Suitable vectors include SV40 derivatives and known bacterial plasmids such as E. coli plasmids col El, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and their Derivatives, plasmids (such as RP4); phage DNAs, such as many derivatives of lambda phage (such as NM989) and other phage DNA (such as M13 and filamentous single-stranded phage DNA); yeast plasmids, such as 2μ plasmid or its derivatives ; vectors for use in eukaryotic cells, such as vectors for use in insect or mammalian cells; vectors derived from a combination of plasmids and phage DNAs, such as plasmids modified to utilize phage DNA or other expression control sequences; etc. .

For example, in a baculovirus expression system, non-fusion transfer vectors such as (but not limited to) pVL941 (BamHI cloning site; Summers), pVL1393 (BamHI, SmaI, XbaI, EcoRI, NotI, XmaIII, BglII and PstI cloning sites; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI and BamHI cloning sites; Summers and Invitrogen), and pBlueBacIII (BamHI, Bgl II, PstI, NcoI and HindIII cloning sites with blue/white recombination screening potential; Invitrogen), where the fusion transfer vector such as (but not limited to) pAc700 (BamHI and KpnI cloning sites, where the BamHI recognition site begins at the start codon; Summers), pAc701 and pAc702 (same as pAc700 but with a different reading frame), pAc360 (the BamHI cloning site is located 36 base pairs downstream of the polyhedrin start codon; Invitrogen ( 195)) and pBlueBacHisA, B, C (three different reading frames, with BamHI, Bgl II, PstI, NcoI and HindIII cloning sites, with N-terminal peptide for ProBond purification, and with blue/white recombination Screening; Invitrogen(220)).

Mammalian expression vectors contemplated for use in the present invention include vectors with an inducible promoter such as the dihydrofolate reductase (DHFR) promoter, such as any expression vector with a DHFR expression vector or a DHFR/methotrexate co-amplification vector, such as pED(PstI, SalI, SbaI, SmaI and EcoRI cloning sites, this vector expresses both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991). Alternatively, Glutamine synthetase/methionine sulfoximine co-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI and BclI cloning sites, wherein the vector expresses glutamine synthetase and the cloned gene; Celltech). In another implementation In the scheme, vectors that direct episomal expression under the regulation of Epstein-Barr virus (EBV) can be used, such as pREP4 (BamHI, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII and KpnI cloning sites, constitutive RSV-LTR promoter , hygromycin selectable marker; Invitrogen), pCEP4 (BamHI, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII and KpnI cloning sites, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen) , pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamHI cloning site, inducible metallothionein Iia gene promoter, hygromycin selectable marker; Invitrogen), pREP8 (BamHI, XhoI, NotI, HindIII, NheI and KpnI cloning sites, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI and BamHI cloning sites, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminal peptide that can be purified by ProBond resin and can be cleaved by enterokinase; Invitrogen). Can be selected for use in the present invention Mammalian expression vectors include pRc/CMV (HindIII, BstXI, NotI, SbaI and ApaI cloning sites, G418 selection; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning sites, G418 selection; Invitrogen )wait. Vaccinia mammalian expression vectors useful in the present invention (see Kaufman, 1991, supra) include, but are not limited to, pSC11 (SmaI cloning site, TK- and β-gal selection), pMJ601 (SalI, SmaI, AflI, NarI , BspMII, BamHI, ApaI, NheI, SacII, KpnI and HindIII cloning sites, TK- and β-gal selection), and pTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamHI and Hpa cloning sites, TK or XPRT option).

Yeast expression systems can also be used in the present invention to express GAVE18 protein, variants, analogs or derivatives thereof. Vectors that can be used in the present invention are mentioned only two vectors, for example the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamHI, SacI, KpnI and HindIII cloning sites; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamHI, SacI, KpnI and HindIII cloning sites, N-terminal peptide purified with ProBond resin and cleavable by enterokinase; Invitrogen).

Once a particular recombinant DNA molecule has been identified and isolated, several methods are known in the art for its propagation. Once a suitable host system and growth conditions have been established, recombinant expression vectors can be propagated and produced in large quantities. As previously explained, expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; phage vectors (e.g., λ phage); and plasmid and cosmid DNA vectors, only some of which are mentioned here.

In addition, host cell strains can be selected that modulate the expression of the inserted sequence or that modify and process the gene product in the specific manner desired. Different host cells have characteristic and specific mechanisms for translational and post-translational processing and modification of proteins (eg glycosylation, cleavage [eg signal sequence]). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein being expressed. For example, expression in bacterial systems can be used to produce non-glycosylated nucleoprotein products.

transgenic animal

The host cells of the invention can also be used to make non-human transgenic animals. For example, in one embodiment, the host cell of the present invention is a fertilized oocyte or embryonic stem cell into which a sequence encoding GAVE18 has been introduced. The host cells can then be used to produce non-human transgenic animals with exogenous GAVE18 sequences introduced into their genomes, or homologously recombined animals in which endogenous GAVE18 sequences have been altered. The animal can be used to study the function and/or activity of GAVE18, and to identify and/or evaluate modulators of GAVE18 activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, wherein one or more cells of the animal comprise a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.

As used herein, the term "transgene" refers to exogenous DNA that is integrated into the genome of the cell from which a transgenic animal develops and that is still present in the genome of the mature animal. The transgene directs expression of the encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombination animal" is a non-human animal, preferably a mammal, more preferably a mouse, whose endogenous GAVE18 gene has been altered by homologous recombination. This is done before the animal develops, between endogenous genes and foreign DNA molecules that are introduced into the animal's cells, such as the animal's embryonic cells.

The transgenic animal of the present invention can use one of the above-mentioned transfection methods to introduce the nucleic acid encoding GAVE18 into the male pronucleus of the fertilized oocyte. The oocytes are then allowed to develop in the pseudopregnant female surrogate animal. A GAVE18 cDNA sequence such as that in (SEQ ID NO: 1) can be introduced as a transgene into the genome of a non-human animal. Alternatively, non-human homologues of the human GAVE18 gene (such as the mouse GAVE18 gene) can be isolated based on hybridization to human GAVE18 cDNA and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of transgene expression. Tissue-specific regulatory sequences can be operably linked to the GAVE18 transgene to direct expression of the GAVE18 protein in specific cells. Methods for producing transgenic animals, particularly animals such as mice, by embryo manipulation and microinjection are routine in the art and can be found, for example, in US Pat. Nos. 4,736,866 and 4,870,009, US Pat. Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods can be used to generate other transgenic animals in which the transgene is present in the genome and/or express GAVE18 mRNA in the tissues or cells of the animal. The starting transgenic animal can be used to breed other animals carrying the transgene. In addition, transgenic animals carrying a transgene encoding GAVE18 can be further bred into transgenic animals carrying other transgenes.

To generate homologous recombination animals, vectors are prepared that contain at least a portion of the GAVE18 gene (e.g., a homologue of the human or non-human GAVE18 gene, such as the mouse GAVE18 gene) into which deletions, additions, or substitutions have been introduced so that the (eg, functional disruption) of the GAVE18 gene. In a preferred embodiment, the vector is designed such that the function of the endogenous GAVE18 gene is disrupted following homologous recombination (ie, no longer encodes a functional protein; also referred to as a "knockout" vector).

Alternatively, the vector can be designed so that the endogenous GAVE18 gene is mutated or altered following homologous recombination but still encodes a functional protein (eg, upstream regulatory regions can be altered thereby altering expression of the endogenous GAVE18 protein).

In this homologous recombination vector, the altered GAVE18 gene portion is surrounded by other nucleic acids of the GAVE18 gene at the 5' and 3' ends, thereby allowing the separation between the exogenous GAVE18 gene carried by the vector and the endogenous GAVE18 gene present in embryonic stem cells. Homologous recombination occurs. The length of this additional flanking GAVE18 nucleic acid should be sufficient to allow successful recombination with the endogenous gene. Typically, several kilobases of flanking DNA (5' and 3' ends) are included in the vector (see, eg, Thomas et al., Cell (1987) 51:503 for a description of homologous recombination vectors).

The vector is introduced (eg, by electroporation) into an embryonic stem cell line, and cells are selected for homologous recombination of the introduced GAVE18 gene with the endogenous GAVE18 gene (see, e.g., Li et al., Cell (1992) 69:915). The selected cells are then injected into blastocysts of animals such as mice to form chimeric aggregates (see, for example, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach), Robertson ed., IRL, Oxford, (1987), pp. 113-152). The chimeric embryos are then implanted into a suitable pseudopregnant female surrogate animal and allowed to grow to term. Progeny with homologously recombined DNA in germ cells can be used to breed animals through which all cells of the bred animal will contain homologously recombined DNA through the germline transmission of the transgene.

Methods for constructing homologous recombination vectors and homologous recombination animals can further be found in Bradley, Current Opinion in Bio/Technology (1991) 2:823-829 and PCT Publication No. WO 90/11354 , WO 91/01140, WO 92/0968 and WO 93/04169 described.

In another embodiment, the resulting transgenic non-human animal may contain a system selected to allow for the regulation of expression of the transgene. An example of such a system is the cre/loxP recombinase system of bacteriophage P1. The cre/loxP recombinase system is described in, eg, Lakso et al., Proc Natl Acad USA (1992) 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorrnan et al., Science (1991) 251:1351-1355). If the cre/loxP recombinase system is to be used to regulate the expression of transgenes, animals containing both the transgene encoding the cre recombinase and the selected protein will be required. Such animals can be provided by the construction of "double" transgenic animals, eg, by mating two transgenic animals, one containing the transgene encoding the selected protein and the other containing the transgene encoding the recombinase.

Clones of the non-human transgenic animals described herein can be prepared according to the methods described in Wilmut et al., Nature (1997) 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669. Briefly, cells of transgenic animals, such as somatic cells, can be isolated and induced to exit the growth cycle and enter the _G0 phase. This quiescent cell is then fused with an enucleated oocyte from the same animal species as the isolated quiescent cell, by application of, for example, electrical pulses. The reconstituted oocytes are then cultured to develop into morulae or blasts, which are then transferred into pseudopregnant female surrogate animals. The offspring of a female surrogate animal are clones of the animal from which the isolated cells (eg, somatic cells) were derived.

pharmaceutical composition

The GAVE18 nucleic acid molecules, GAVE18 proteins and anti-GAVE18 antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. The composition generally comprises the nucleic acid molecule, protein or antibody, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, excipients, carriers, diluents, coatings, antibacterial and antifungal agents, isotonic and antifungal agents suitable for pharmaceutical administration. Delayed absorption agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agents are contemplated for use in the compositions, except those incompatible with the active compounds. Supplementary active compounds can also be incorporated into the compositions.

The pharmaceutical compositions of the invention are formulated so as to be suitable for the intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal and subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal and rectal. Solutions or suspensions for parenteral, intradermal and subcutaneous administration may contain the following ingredients: sterile diluent (such as water for injection), saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or Other synthetic solvents; antimicrobials such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate or phosphate and for adjusting osmotic Pressurized reagents such as sodium chloride or dextrose. Adjustment of pH can be performed with acids or bases such as HCl or NaOH. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials (made of glass or plastic) for storage.

Pharmaceutical compositions suitable for injectable administration include sterile aqueous solutions (water-miscible) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, "Cremophor EL" (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be It is a fluid that is easy to inject. The composition must be stable under production and storage conditions, and must avoid the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or a dispersion medium, containing such as water, ethanol, polyols (such as glycerol, propylene glycol) and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained by, for example, the use of coating materials (such as lecithin), the maintenance of the required particle size in the case of dispersions and the application of surfactants A variety of antibacterial and antifungal agents are available to prevent the action of microorganisms, such as parabens, chlorobutanol, phenol, ascorbic acid, sodium ethylmercury thiosalicylate, etc. In many cases, the combination Isotonic agents are preferably included in the composition, such as sugar, polyalcohol (such as mannitol, sorbitol) or sodium chloride. The delayed absorption of the injectable composition can be realized by adding an agent that delays absorption in the composition, said Reagents such as aluminum monostearate and gelatin.

The preparation of sterile injectable solutions can be carried out by incorporating the active compound (such as GAVE18 protein or anti-GAVE18 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients listed above (as required) , then filter sterilized. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder containing the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. The composition can be sealed in a gelatin capsule or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated into excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally, swished and expectorated or swallowed.

Pharmaceutically compatible binders and/or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or Orange dressing. For inhalation administration, the compounds are delivered in the form of an aerosol spray which can be generated from a pressurized container or dispenser or nebuliser containing a suitable propellant (eg, a gas such as carbon dioxide).

Systemic administration can also be by transdermal or transmucosal routes. For transdermal or transmucosal administration, penetrants suitable for the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished by application of nasal sprays or suppositories. For transdermal administration, the active compound formulations may be in the form of ointments, salves, gels or creams, as known in the art.

The compounds may also be prepared for rectal delivery in the form of suppositories (eg, using conventional suppository bases with lubricating materials, such as cocoa butter or other glycerides, which melt at the body temperature of the mammal) or enema retentions.

In a specific embodiment, the active compounds are formulated with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

Methods for preparation of such formulations will be apparent to those skilled in the art. Materials are also available from suppliers such as Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes with monoclonal antibodies targeted to infected cells) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, eg as described in US Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form, which facilitates administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suited as a whole for the patient to be treated; each unit containing a predetermined quantity of active compound calculated to produce, in association with the required pharmaceutical carrier, produce the desired therapeutic effect. Depending on the type and severity of the disease, an initial candidate dose of about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of the antibody is administered to the patient, either by, for example, one or more separate administrations or by continuous infusion. Note. Depending on the factors mentioned above, typical daily doses may range from about 1 μg/kg to 100 mg/kg or greater. For repeated administrations over several days or longer, depending on the disease, treatment may continue until the Desired suppression of disease symptoms. However, other dosing regimens can also be used. The progress of treatment can be easily monitored by conventional techniques and tests. An exemplary agent dosing regimen is disclosed in WO 94/04188. The specification for the unit dosage forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound, the particular therapeutic effect to be obtained and the limitations inherent in the art of compounding the active compound for use in individual therapy.

Furthermore, the nucleic acid molecules of the present invention can be inserted into vectors and used as gene therapy vectors. Delivery of gene therapy vectors to patients can be accomplished by, for example, intravenous injection, topical administration (U.S. Pat. No. 5,328,470), or by stereotaxic injection (see, e.g., Chen et al., Proc Natl Acad USA (1994) 91:3054- 3057) to proceed. Pharmaceutical formulations of gene therapy vectors may comprise the gene therapy vector in an acceptable diluent; or may comprise a sustained release matrix with the gene delivery means embedded therein. Alternatively, when the entire gene delivery vector can be produced intact from a recombinant cell, such as a retroviral vector, the pharmaceutical formulation can include one or more cells producing the gene delivery system.

The pharmaceutical composition can be included in a container, pack or dispenser together with instructions for administration.

Applications and methods of the invention

The nucleic acid molecules, proteins, protein homologues and antibodies of the invention may be used in one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosome mapping, tissue typing, forensic biology); c) predictive medicine (eg, diagnostic tests, prognostic tests, clinical monitoring tests, and pharmacogenomics); and d) therapeutic approaches (eg, therapeutic and preventive). GAVE18 proteins interact with other cellular proteins and thus can be used to (i) regulate cell proliferation; (ii) regulate cell differentiation; and (iii) regulate cell survival. The isolated nucleic acid molecules of the present invention can be used to express GAVE18 protein (for example, in a gene therapy application in a host cell by a recombinant expression vector), to detect GAVE18 mRNA (such as in a biological sample) or to detect GAVE18 gene genetic damage and for modulating GAVE18 activity. In addition, GAVE18 proteins can be used to screen for drugs or compounds that modulate GAVE18 activity or expression; and to treat disorders characterized by under- or over-production of GAVE18 protein, or in forms of GAVE18 protein produced compared to GAVE18 wild-type protein Reduced or abnormal activity. In addition, the anti-GAVE18 antibodies of the present invention can be used to detect and isolate GAVE18 protein, and to modulate GAVE18 activity. The invention further relates to novel agents identified by the screening assays described above, and their use in the treatments described herein.

screening test

Activation of the G protein receptor in the presence of endogenous ligand will allow the formation of a G protein receptor complex, thereby resulting in binding of GTP to the G protein. The GTPase domain of the G protein can slowly hydrolyze GTP to GDP, leading to receptor inactivation under normal conditions. But constitutively activated receptors continue to convert GDP to GTP.

[ ³⁵ S]GTPyS, a non-hydrolyzable substrate of G protein, can be used to monitor the enhanced binding of G protein to membranes expressing constitutively activated receptors. Traynor and Nahorski reported that [ ³⁵ S]GTPyS can be used to monitor membrane-coupled G proteins in the presence and absence of ligand (Mol Pharmacol (1995) 47(4):848-54). This assay system is preferably used for the initial screening of candidate compounds because the system can be used universally for all G protein-coupled receptors, regardless of the type of specific G protein that binds to the receptor.

G _s20 stimulates adenylyl cyclase, whereas G _i and G _o inhibit this enzyme. As is well known in the art, adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, a constitutively active GPCR coupled to a _Gs protein would correlate with increased cellular levels of cAMP. Alternatively, constitutively activated GCPRs coupled to G _i (or G _o ) proteins would be associated with reduced cellular levels of cAMP, see "Indirect Mechanism of Synaptic Transmission", Chapter 8, From Neurons to Brain (Third Edition), edited by Nichols et al., Sinauer Associates, Inc., 1992. Therefore, assays to detect cAMP can be used to determine whether a candidate compound is an inverse agonist of the receptor. Various methods for measuring cAMP known in the art can be used. In one embodiment, anti-cAMP antibodies are used in ELISA-based assays. In another embodiment, whole cell second messenger reporter systems are contemplated (see PCT Publication No. WO 00/22131).

In a related aspect, cyclic AMP drives gene expression by promoting the binding of cAMP-responsive DNA-binding proteins or transcription factors (CREB), which then act on specific The site binds to the promoter and drives gene expression. Reporter systems can therefore be constructed with a promoter comprising multiple cAMP response elements preceding a reporter gene such as β-galactosidase or luciferase. Further, when constitutively activated Gs-linked receptors lead to accumulation of cAMP, the gene can be activated and the reporter protein expressed. Reporter proteins such as beta-galactosidase or luciferase can then be detected using standard biochemical assays (PCT Publication No. WO 00/22131).

Other G proteins, such as G _o and G _q , are associated with the activation of phospholipase C, which in turn hydrolyzes the phospholipid PIP2 to release two intracellular messengers: diacylglycerol (DAG) and 1,4,5-tris Phosphoinositides (IP3). Increased IP3 accumulation correlates with activation of _Gq -associated receptors and _Go -associated receptors (PCT Publication No. WO 00/22131). Assays to detect accumulation of IP3 can be used to determine whether a candidate compound is an inverse agonist of a _Gq -related receptor or a _Go -related receptor. _Gq -associated receptors can also be detected using the AP1 reporter assay, which detects whether _Gq -dependent phospholipase C causes activation of genes containing AP1 elements. Thus, activated _Gq -related receptors will show an increase in the expression of that gene, whereas an inverse agonist will show a decrease in that expression.

The present invention also provides methods (herein also referred to as "screening assays") for identifying modulators, i.e. candidates or tests that bind to the GAVE18 protein or have a stimulatory or inhibitory effect on, for example, GAVE18 expression or GAVE18 activity Compounds or agents (eg, peptides, peptidomimetics, small molecules, or other drugs).

In one embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of membrane-bound forms of GAVE18 proteins, polypeptides, or biologically active portions thereof. Test compounds of the invention can be obtained using any of a number of means in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid-phase or liquid-phase libraries; libraries requiring deconvolution Synthetic library methods; "single bead single compound" library methods; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four methods are applicable to peptide, non-peptide oligomer, or small molecule compound libraries (Lam, Anticancer Drug Des (1997) 12:145).

Examples of methods for synthesizing molecular libraries can be found in the art, e.g., DeWitt et al., Proc Natl Acad USA (1993) 90:6909; Erb et al., Proc Natl Acad USA (1993) 90:6909; Acad USA) (1994) 91:11422; Zuckermann et al., J Med Chem (1994) 37:2678; Cho et al., Science (1993) 261:1303; Carrell et al., Angew Chem Int Ed Engl (1994) 33:2059; Carell et al., Angew Chem Int Ed Engl (1994) 33:2061 and Gallop et al., JMed Chem (1994) 37:1233.

Compound libraries can be presented in solution (e.g., Houghten Bio/Techniques (1992) 13:412-421 ) or on beads (Lam, Nature (1991) 354:82-84 ), on a chip (Fodor, Nature ( Nature) (1993) 364:555-556), or bacteria (US Patent No. 5,223,409), spores (US Patent Nos. 5,571,698; 5,403,484 and 5,223,409), plasmids (Cull et al., Proc Natl Acad USA) ( 1992) 89:1865-1869) or on phages (Scott et al., Science (1990) 249:386-390; Devlin, Science (1990) (1990) 249:404-406; Cwirla et al., US National Academy of Sciences (Proc Natl Acad USA) (1990) 87:6378-6382; and Felici, J Mol Biol (1991) 222:301-310).

In one embodiment, the assay is a cell-based assay in which cells expressing a membrane-bound form of the GAVE18 protein (or a biologically active portion thereof) on the cell surface are contacted with a test compound and the ability of the test compound to bind the GAVE18 protein is determined. For example, the cells may be yeast cells or cells of mammalian origin. The determination of the binding ability of the test compound to the GAVE18 protein can be carried out, for example, by coupling the test compound to a radioactive isotope or enzyme label, such that the binding of the test compound to the GAVE18 protein or a biologically active portion thereof can be detected by the label in the complex. Compounds are identified. For example, the test compound can be directly or indirectly labeled125I, ^35S , ^{14C or3H} and the ^detection of the radioisotope can be by direct counting of radioactivity or by scintillation counting. Alternatively, test compounds can be enzymatically labeled using, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and detection of enzymatic labeling can be accomplished by measuring the conversion of the appropriate substrate to product. In a preferred embodiment, the assay comprises contacting a cell expressing a membrane-bound form of the GAVE18 protein or a biologically active portion thereof on the cell surface with a compound known to bind GAVE18 to form a test mixture, contacting the test mixture with the test compound and The ability of the test compound to interact with the GAVE18 protein is determined, wherein determining the ability of the test compound to interact with the GAVE18 protein comprises determining the ability of the test compound to bind preferentially to GAVE18 or a biologically active portion thereof compared to known compounds.

In another embodiment, the assay is a cell-based assay comprising contacting cells expressing a membrane-bound form of the GAVE18 protein, or a biologically active portion thereof, on the cell surface with a test compound, and determining that the test compound modulates (e.g. stimulates or inhibits) the GAVE18 protein or the activity of its biologically active portion. The ability of a test compound to modulate the activity of GAVE18 or a biologically active portion thereof can be determined, for example, by assaying the ability of a GAVE18 protein to bind or interact with a GAVE18 target molecule. As used herein, "target molecule" refers to a molecule that naturally binds or interacts with a GAVE18 protein, such as a molecule on the surface of a cell expressing the GAVE18 protein, a molecule on the surface of a second cell, a molecule in the extracellular peripheral region, and A molecule associated with the inner surface of a cell membrane or a cytoplasmic molecule. The GAVE18 target molecule may not be the GAVE18 molecule or the GAVE18 protein or polypeptide of the present invention. In one embodiment, the GAVE18 target molecule is a component of a signal transduction pathway that facilitates the transduction of an extracellular signal (eg, a signal produced by a compound binding a membrane-bound form of the GAVE18 molecule) across the membrane into the cell. For example, the target molecule can be a second intracellular protein with catalytic activity or a protein that facilitates the association of downstream signaling molecules with GAVE18.

The ability of a GAVE18 protein to bind or interact with a GAVE18 target molecule can be determined by one of the methods described above for determining direct binding. In preferred embodiments, the ability of a GAVE18 protein to bind or interact with a GAVE18 target molecule can be determined by measuring the activity of the target molecule. Methods for measuring the activity of target molecules include, for example: detecting the induction of target molecules on intracellular second messengers (such as intracellular Ca ²⁺ , diglyceride, IP3, etc.), detecting the catalytic/enzymatic activity of target molecules on suitable substrates, Detecting induction of a reporter gene such as a GAVE18 effector regulatory element operably linked to a nucleic acid encoding a detectable marker such as luciferase or detecting a cellular response such as cell differentiation or cell proliferation.

The invention also extends to cell-free assays comprising contacting the GAVE18 protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind the GAVE18 protein or biologically active portion thereof. Binding of test compounds to GAVE18 protein can be detected directly or indirectly as described above. In a preferred embodiment, the assay comprises contacting the GAVE18 protein or a biologically active portion thereof with a compound known to bind GAVE18 to form a test mixture, contacting the test mixture with the test compound and determining the ability of the test compound to interact with the GAVE18 protein, Wherein the determination of the ability of the test compound to interact with the GAVE18 protein comprises determining the ability of the test compound to preferentially bind to GAVE18 or a biologically active portion thereof compared to known compounds.

Another cell-free assay of the invention involves contacting a GAVE18 protein or a biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of the GAVE18 protein or a biologically active portion thereof. Determination of the ability of a test compound to modulate the activity of GAVE18 can be achieved, for example, by determining the ability of a GAVE18 protein to bind a GAVE18 target molecule using one of the methods described above for determining direct binding. In an alternative embodiment, the ability of a test compound to modulate GAVE18 activity can be determined by determining the ability of the GAVE18 protein to further modulate GAVE18 target molecules. For example, the catalytic/enzymatic activity of a target molecule towards an appropriate substrate can be assayed as described above.

Yet another cell-free assay of the present invention comprises contacting the GAVE18 protein or a biologically active portion thereof with a known compound capable of binding GAVE18 to form a test mixture, contacting the test mixture with the test compound and determining the ability of the test compound to interact with the GAVE18 protein , wherein the determination of the ability of the test compound to interact with the GAVE18 protein comprises determining the ability of the GAVE18 protein to preferentially bind to or modulate the activity of the GAVE18 target molecule.

Receptors can be activated by non-ligand molecules that do not necessarily inhibit ligand binding but can cause structural changes in the receptor such that G protein binding or, possibly receptor aggregation, dimerization or clustering, thereby cause activation. For example, antibodies can be raised from multiple sites of GAVE18 exposed on the cell surface. These antibodies activate cells through the G protein cascade, which can be determined by standard assays such as monitoring cAMP levels or intracellular Ca2 ⁺ levels. Monoclonal antibodies may be preferred as they involve molecular mapping, especially epitope mapping. Monoclonal antibodies can be raised from intact receptors expressed on the cell surface as well as from peptides known to form on the cell surface. The method of Geysen et al., US Patent No. 5,998,577 can be used to obtain large numbers of related peptides. Antibodies found to activate GAVE18 may be modified to minimize activities unrelated to activation of GAVE18, such as complement fixation. Thus, truncations or mutations can be performed on antibody molecules to minimize or abolish activities other than activating GAVE18. For example, for some antibodies, only the antigen binding portion is required. In this way, the Fc portion of the antibody can be removed.

Cells expressing GAVE18 were exposed to antibodies to activate GAVE18. The activated cells are then exposed to a variety of molecules in order to identify those that alter the activity of the receptor, either increasing or decreasing the level of activation. Molecules for this purpose can then be tested in the absence of antibodies on cells expressing GAVE18 to observe the effect on non-activated cells. Target molecules can then be detected and modified into drug candidates using well-known techniques for the treatment of disorders associated with altered GAVE18 metabolism.

The cell-free assays of the present invention are suitable for use with both soluble and membrane-bound forms of GAVE18. For cell-free assays involving the membrane-bound form of GAVE18, it may be necessary to utilize solubilizing agents to maintain the membrane-bound form of GAVE18 in solution. Examples of the solubilizer include nonionic detergents such as n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N- -Methyl Glucamide, Triton X-100, Triton X-114, ^Thesit® , Isodecyl Poly(ethylene glycol ether)n, 3-[(3-Chloroaminopropyl)dimethylammonium]- 1-propanesulfonic acid (CHAPS), 3-[(3-chloroaminopropyl)dimethylammonium]-2-hydroxy-1-propanesulfonic acid (CHAPSO) or N-dodecyl-N,N- Dimethyl-3-ammonium-1-propanesulfonic acid.

In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize GAVE18 or its target molecules to allow easy separation of the complexed form from the non-complexed form of one or both of said proteins, and to accommodate automation of the assay. Binding of a test compound to GAVE18 or interaction of GAVE18 to a target molecule in the presence or absence of a candidate compound can be accomplished in any vessel suitable for holding the reactants. Examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein may be provided with the addition of a domain that allows one or both of the above proteins to bind to the matrix. For example, a glutathione-S-transferase/GAVE18 fusion protein or a glutathione-S-transferase/target fusion protein can be adsorbed to glutathione SEPHAROSE beads (Sigma Chemical, St. Louis, MO). Alternatively, glutathione-derivatized microtiter plates can be used, and the microtiter plate is then exposed to the test compound, or the test compound and the non-adsorbed target protein or GAVE18 protein, and the mixture is incubated under conditions that favor complex formation (e.g. under physiological salt and pH conditions). Following incubation, the beads or wells of the microtiter plate are washed to remove any unbound components, and complex formation is assayed, either directly or indirectly, see above. Alternatively, the complex can be dissociated from the matrix and the level of GAVE18 binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the present invention. For example, GAVE18 or its target molecules can be immobilized using biotin or streptavidin. Biotinylated GAVE18 or target molecules can be produced from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kits, Pierce Chemicals, Rockford, IL), and Immobilization was performed in wells of streptavidin-coated 96-well plates (Pierce Chemicals). Alternatively, the wells of the plate can be derivatized with an antibody that reacts with GAVE18 or the target molecule but does not block the binding of the GAVE18 protein to the target molecule, whereby unbound target or GAVE18 is captured within the wells by antibody binding. In addition to the methods described above for the detection of GST-immobilized complexes, methods for detecting the complexes include immunodetection of the complexes using antibodies reactive with GAVE18 or the target molecule and relying on the detection of complexes with GAVE18 or target molecules. Enzyme-linked assays for the enzymatic activity of molecular linkages.

In another embodiment, modulators of GAVE18 expression can be identified by a method wherein cells are contacted with a candidate compound and intracellular GAVE18 mRNA or protein expression is determined. The expression level of GAVE18 mRNA or protein in the presence of the candidate compound is compared to the expression level of GAVE18 mRNA or protein in the absence of the candidate compound. Then, based on this comparison, one can identify whether a candidate compound is a modulator of GAVE18 expression. For example, a candidate compound is identified as a stimulator or agonist of GAVE18 mRNA or protein expression when the expression of GAVE18 mRNA or protein in the presence of the candidate compound is greater (statistically significantly greater than) the expression in the absence of the compound. Alternatively, a candidate compound is identified as an inhibitor or antagonist of GAVE18 mRNA or protein expression when the expression of GAVE18 mRNA or protein in the presence of the candidate compound is lower (statistically significantly lower) than in the absence of the compound. Candidate compounds were identified as inverse agonists if GAVE18 activity was reduced in the presence of ligand or agonist, or below baseline in the case of constitutive GAVE18. Intracellular GAVE18 mRNA or protein expression levels can be determined by the methods described herein for the detection of GAVE18 mRNA or protein.

In yet another aspect of the invention, the GAVE18 protein can be used as a "bait protein" in a two-hybrid or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell (1993) 72:223-232; Madura etc., J Biol Chem (1993) 268: 12046-12054; Barrel et al., Bio/Techniques (1993) 14: 920-924; Iwabuchi et al., Oncogene (1993) 8: 1693-1696 and PCT Publication No. WO 94/10300), to identify other proteins that bind or act on GAVE18 ("GAVE18-binding protein" or "GAVE18-bp") and regulate GAVE18 activity. The GAVE18-binding protein may also be involved in signal transmission through the GAVE18 protein as an upstream or downstream element of eg the GAVE18 pathway.

Since a large amount of pure GAVE18 can be prepared, the conformational physical characteristics of possible functional regions can be determined for rational drug design. For example, the IC3 region and the EC domain of the molecule are regions of particular interest. Once the shape and ionic configuration of the regions are determined, drug candidates that can interact with these regions can be shaped and then tested in intact cells, animals and patients. Methods that can obtain this 3-D structural information include X-ray crystallography, NMR spectroscopy, molecular modeling, and the like. The 3-D structure can also lead to the identification of similar conformational sites in other known proteins for which known drugs already exist to act at this site. These drugs or their derivatives may be useful for GAVE18.

The invention also relates to novel agents identified by the above screening assays, and their use in the treatments described herein.

Test of the invention

A. Detection test

Portions or fragments of the DNA sequences of the invention can be used as polynucleotide reagents in a variety of ways. For example, sequences can be used to: (i) map corresponding genes on chromosomes and thereby locate gene regions associated with genetic diseases; (ii) identify individuals from small biological samples (tissue typing); and (iii) Assist in the forensic identification of biological samples. These applications are described in the following subsections.

1. Chromosome mapping

Once the sequence of the gene (or a portion of the sequence) is isolated, the sequence can be used to map the GAVE18 gene on the chromosome. Accordingly, the GAVE18 nucleic acid molecules described herein, or fragments thereof, can be used to map GAVE18 in the genome. Mapping the GAVE18 sequence in the genome, especially the human genome, is an important first step in determining the relationship between the sequence and disease-related genes. Briefly, the GAVE18 gene can be mapped in the genome by preparing PCR primers (preferably 15-25 bp in length) from the GAVE18 sequence. The primers can be used for PCR screening of somatic cell hybrids containing a single human chromosome. Only those hybrids containing the human gene corresponding to the GAVE18 sequence produced amplified fragments.

Somatic cell hybrids are prepared by fusing somatic cells of different mammalian origin (eg, cells from humans and mice). When hybrids of human and mouse cells grow and divide, human chromosomes are randomly lost, while mouse chromosomes are retained. By using a medium in which mouse cells (due to lack of a specific enzyme) cannot but human cells can grow, a human chromosome containing the gene encoding the desired enzyme will be preserved. By using multiple media, a panel of heterozygous cell lines can be established. Each cell line in the group contains a single human chromosome or a small number of human chromosomes and a complete set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes (D'Eustachio et al., Science ) (1983) 220:919-924). Somatic hybrids containing only segments of human chromosomes can also be prepared by using human chromosomes with translocations and deletions.

PCR mapping of somatic heterozygotes is a rapid method to localize specific sequences on specific chromosomes. Three or more sequences can be positioned per thermal cycler per day.

Other mapping strategies can likewise be used to map GAVE18 sequences on specific chromosomes in the genome, including in situ hybridization (described in Fan et al., Proc Natl Acad USA (1990) 87:6223-27) , Chromosome prescreening with marker flow sorting and preselection by hybridization to chromosome-specific cDNA libraries.

Precise chromosomal localization can be provided in one step using fluorescence in situ hybridization (FISH) of DNA sequences and metaphase chromosome spreads. Chromosomal spreads can be prepared using cells arrested in metaphase by chemicals such as colchicine, which disrupts the mitotic spindle. Chromosomes can be briefly trypsinized and then stained with Giemsa. Individual chromosomes are identified by a pattern of light and dark bands on each chromosome. FISH can be performed with DNA sequences of 500 or 600 bases. However, clones larger than 1,000 bases are more likely to bind unique chromosomal locations and generate sufficient signal strength for simple detection. Preferably 1,000 bases and more preferably 2,000 bases will be sufficient to produce good results in a reasonable amount of time. See Verma et al. for a review of this technique (Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988)). Chromosome mapping can be inferred in silico considering statistical factors such as log odds scores or mere proximity.

Reagents for chromosome mapping can be applied individually to map a single locus on a chromosome. Also, a set of reagents can be used to label multiple loci and/or multiple chromosomes. For mapping purposes, reagents corresponding to the flanking regions of the GAVE18 gene are actually preferred. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross-hybridization in chromosome mapping.

Once the sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be linked to genetic map data (this data is deposited, for example, in McKusick, Mendelian Inheritance in Man, available online from Welch Medical Library, Johns Hopkins University). Relationships between genes and diseases mapped to the same chromosomal region can then be determined by linkage analysis (co-inheritance of physically adjacent genes), eg see Egeland et al., Nature (1987) 325:783-787 in the description.

In addition, differences in DNA sequence between individuals affected or not affected by a GAVE18-associated disease can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, the mutation may be causative for a particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural changes in chromosomes, such as deletions or translocations, that can be observed in chromosome spreads or can be detected using PCR based on the DNA sequence . Ultimately, genes from several individuals can be fully sequenced to confirm the presence of mutations and to distinguish mutations from polymorphisms.

2. Tissue classification

The GAVE18 sequence of the present invention can also be used to identify individuals starting from a small number of biological samples. For example, the US military is considering restriction fragment length polymorphism (RFLP) for personnel identification. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed in a Southern blot to generate unique bands for identification. This method avoids the limitations of current identification tags (Dog Tags) methods, which can be lost, converted or stolen, which makes positive identification difficult. The sequences of the invention can be used as additional DNA markers for RFLP (described in US Patent No. 5,272,057).

In addition, the sequences of the invention can be used to provide an alternative technique to base-by-base determination of the actual DNA sequence of selected locations in an individual's genome. Thus, the GAVE18 sequence described here can be used to prepare two stretches of PCR primers targeting the 5' and 3' ends of the sequence. The primers can then be used to amplify the individual's DNA and subsequently provide its sequence.

The corresponding set of DNA sequences obtained from an individual in this way will provide a unique means of identifying the individual, since each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and organizations. The GAVE18 sequence of the present invention is a unique part of the human genome. There is some degree of allelic variation in the coding regions of the sequence and a higher degree of variation in the noncoding regions. It is estimated that the frequency of allelic variation among human individuals is approximately once every 500 bases. Each of the sequences described herein can be used to some extent as a standard, compared to DNA from an individual for identification purposes. Due to the greater number of polymorphisms in non-coding regions, fewer sequences are required to distinguish individuals. Using a set of approximately 10 to 1,000 primers (each primer yielding 100 bases of amplified non-coding sequence), the non-coding sequence of SEQ ID NO: 1 provides positive identification of the individual. If using predicted coding sequences such as those in SEQ ID NO: 1, a more suitable number of primers would be 500-2,000 for positive individual identification.

If a set of reagents from the GAVE18 sequence (as described herein) is used to generate a database of unique identifiers for an individual, the same reagents can then be used to identify tissue from that individual. Using this unique identification database, positive identification of individuals (living or dead) can be achieved starting from an extremely small number of tissue samples.

3. Application of partial sequence of GAVE18 in forensic biology

DNA-based identification techniques can also be used in forensic biology. Forensic biology is the field of science that applies genetic typing of biological evidence found at crime scenes as a means of positively identifying (eg, perpetrators). For identification, PCR techniques can be used to amplify DNA sequences from small biological samples, such as tissue (such as hair or skin) or bodily fluids (such as blood, saliva or semen) found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing the source of the biological sample to be identified.

The sequences of the present invention can be used to provide polynucleotide reagents such as PCR primers targeting specific sites in the human genome, which can increase the reliability of forensic identification based on DNA. For example, a nucleic acid of interest may provide another "identifying marker" (ie, another DNA sequence unique to a particular individual). As mentioned above, actual base sequence information can be used for identification purposes as an accurate alternative to fragment patterns generated by restriction enzymes. Sequences targeting the non-coding region of SEQ ID NO: 1 are particularly suitable for this use because of the high number of polymorphisms present in this non-coding region, thereby increasing the discriminatory power to distinguish individuals using this technique. Examples of polynucleotide agents include the GAVE18 sequence or a portion thereof, such as a fragment from the noncoding region of SEQ ID NO: 1 that is at least 20 to 30 bases in length.

The GAVE18 sequences described herein can also be used to provide polynucleotide reagents, such as labeled probes or labeled probes, which can be used, for example, in situ hybridization techniques to identify specific tissues (eg, brain tissue). This is useful when providing a forensic pathologist with cells of unknown origin or degraded tissue. The GAVE18 probe set can be used to identify the species and/or organ type of a tissue.

In a similar manner, reagents such as GAVE18 primers or probes can be used to screen for contaminated tissue cultures (ie to discriminate in culture for the presence of a mixture of different cell types).

B. Predictive Medicine

The invention also relates to the field of predictive medicine, where diagnostic tests, prognostic tests, pharmacogenomics and clinical monitoring tests are used for prognostic (predictive) purposes to preventively treat individuals. Accordingly, one aspect of the present invention relates to a diagnostic assay for determining the expression of GAVE18 protein and/or nucleic acid and the activity of GAVE18 in the context of biological samples such as blood, urine, feces, sputum, serum, cells and tissues. This assay can be used to determine whether an individual has or is at risk of developing a disease or disorder associated with aberrant GAVE18 expression or activity.

The present invention also provides prognostic (or predictive) assays to determine whether an individual is at risk for a disorder associated with GAVE18 protein, nucleic acid expression or activity. For example, biological samples can be tested for mutations in the GAVE18 gene. The assay can be used for prognostic or predictive purposes whereby an individual can be treated prophylactically prior to the onset of a disorder characterized by or associated with the expression or activity of GAVE18 protein, nucleic acid.

Another aspect of the present invention provides methods for determining the expression of GAVE18 protein, nucleic acid, or GAVE18 activity in an individual to thereby select an appropriate therapeutic or prophylactic agent for that individual (referred to herein as "pharmacogenomics" ). Pharmacogenomics allows the selection of agents (eg, drugs) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (eg, examining an individual's genotype to determine the individual's ability to respond to a particular agent).

Yet another aspect of the present invention involves monitoring the effect of agents (such as drugs or other compounds) on the expression or activity of GAVE18 in clinical trials. These and other agents are described in further detail in the following sections.

1. Diagnostic tests

Exemplary methods for detecting the presence or absence of GAVE18 in a biological sample include: obtaining a biological sample from a subject, and combining the biological sample with a compound that can detect a GAVE18 protein or a nucleic acid (such as mRNA or genomic DNA) encoding a GAVE18 protein or Reagent exposure to detect the presence of GAVE18 in biological samples. A preferred reagent for detecting GAVE18 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to GAVE18 mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length GAVE18 nucleic acid, such as a nucleic acid of SEQ ID NO: 1 or a portion thereof, such as at least 15, 30, 50, 100, 250, 500 or more nucleotides in length and sufficient to Oligonucleotides under stringent conditions sufficient to specifically hybridize to GAVE18 mRNA or genomic DNA. Other suitable probes that may be used in the diagnostic assays of the invention are described herein.

A preferred reagent for detecting GAVE18 protein is an antibody capable of binding to GAVE18 protein, preferably a detectably labeled antibody. Antibodies can be polyclonal or, more preferably, monoclonal. Whole antibodies or fragments thereof (eg _Fab or F _(ab')2 ) can be used. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present in the body of a subject. That is, the detection method of the present invention can be used to detect GAVE18 mRNA, protein or genomic DNA in biological samples in vitro and in vivo. For example, techniques for detecting GAVE18 mRNA in vitro include Northern hybridization and in situ hybridization. Techniques used to detect GAVE18 protein in vitro include ELISA, Western blotting, immunoprecipitation, and immunofluorescence. Techniques for in vitro detection of GAVE18 genomic DNA include Southern hybridization. Additionally, techniques for detecting GAVE18 protein in vivo include introducing a labeled anti-GAVE18 antibody into a subject. For example, the antibody can be labeled with a radioactive marker, the presence and location of which in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules from the subject. Alternatively, the biological sample may contain mRNA molecules from the subject or genomic DNA molecules from the subject. A preferred biological sample is a sample of peripheral blood leukocytes isolated from a subject by conventional means.

Therefore, when developing prognostic or diagnostic assays, it may be beneficial to identify nucleic acid or protein polymorphisms associated with disease for the diagnosis of carriers or patients. For example, for rheumatoid arthritis, asthma, Crohn's disease, etc., it would be beneficial to perform a prognostic or diagnostic test. GAVE18 expression levels are elevated in cells in an activated or inflammatory state. Disorders related to inflammation include allergic diseases, colitis, Crohn's disease, edema, contact allergy, anaphylaxis, other forms of arthritis, meningitis and other diseases in which the immune system reacts to damage, so The reaction is causing swelling at a certain site through vasodilation, heat generation, cell aggregation, body fluid, and the like. Therefore, the disturbance of GAVE18 metabolism can be used in the diagnosis of rheumatoid arthritis. Also, molecular mechanisms of rheumatoid arthritis may be detectable, for example, there may be diagnostic SNPs, RFLPs, variability in expression levels, variability in function, etc. that can be detected in tissue samples (eg, blood samples) wait.

In another embodiment, the methods further comprise: obtaining a biological sample from a control subject; contacting the control sample with a compound or reagent capable of detecting GAVE18 protein, mRNA or genomic DNA, so as to detect GAVE protein, mRNA or the presence and amount of genomic DNA; and comparing the presence and amount of GAVE18 protein, mRNA or genomic DNA in the control sample to the presence and amount of GAVE18 protein, mRNA or genomic DNA in the test sample.

High Throughput Identification of Chemical Libraries

Any approach to the identification of compounds that modulate GAVE18 activity can employ high throughput screening. High-pass screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc., Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate all steps, including pipetting of all samples and reagents, liquid dispensing, timed incubations, and final microplate readings, within a detector suitable for the assay. These assembly systems provide high throughput and fast start-up, and offer a high degree of flexibility and customization. Manufacturers of such systems offer several high-throughput, detailed methods. Thus, for example, Zymark Corp. provides a technical bulletin describing a screening system for detecting modulation of gene transcription, ligand binding, and the like.

Reagent test kit

The present invention also includes a kit for detecting the presence of GAVE18 in a biological sample (test sample). The kit can be used to determine whether the subject suffers from or has an increased risk of suffering from a disease (such as malignant tumor) related to the abnormal expression of GAVE18. For example, the kit may comprise a labeled compound or reagent capable of detecting GAVE18 protein or mRNA in a biological sample and means for detecting the amount of GAVE18 in the sample (for example, an anti-GAVE18 antibody or an encoding DNA capable of binding to GAVE18 (such as SEQ ID NO : 1) bound oligonucleotide probe). When the amount of GAVE18 protein or mRNA is higher or lower than the normal level, the kit can also be used to determine whether the subject suffers from or is at risk of suffering from a disease related to the abnormal expression of GAVE18.

For antibody-based kits, it can comprise, for example: (1) a primary antibody that can bind to the GAVE18 protein (e.g., attached to a solid support); and, optionally, (2) that can bind to the GAVE18 protein or to The first antibody binds and is conjugated to a different second antibody with a detectable reagent. If the secondary antibody is not present, another molecule that binds to the primary antibody and can be labeled can be used, or the primary antibody can be labeled. A labeled binding moiety should however be included to serve as a detectable reporter, as is known in the art.

For oligonucleotide-based kits, it may comprise, for example: (1) an oligonucleotide that can hybridize to a GAVE18 nucleic acid sequence, such as a detectably labeled oligonucleotide or (2) an oligonucleotide that can be used to amplify a GAVE18 nucleic acid Molecular primer pairs.

Kits may also contain, for example, buffers, preservatives or protein stabilizers. The kit may also contain the components necessary for the detection of a detectable reagent (eg, an enzyme or a substrate). Furthermore, the kit may also contain a control sample or series of control samples that can be analyzed and compared with the test sample. Each component of the kit is usually contained in a different container, and all these different containers are placed in one package. The package may also include instructions for observing whether the subject suffers from or is at risk of suffering from a disease related to abnormal expression of GAVE18.

2. Prognosis experiment

The methods described herein can further be used as diagnostic or prognostic assays to identify whether a subject has or is at risk of having a disease or condition associated with aberrant GAVE18 expression or activity. For example, assays described herein, such as prospective diagnostic assays or follow-up assays, can be used to identify subjects who have, or are at risk of having, a disorder associated with the expression or activity of a GAVE18 protein, nucleic acid. For example, recent exposure to bacteria or suffering from inflammation associated with asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis are easily checked with this test. Alternatively, prognostic assays can be used to identify subjects who have or are at risk of having the disease or condition.

Accordingly, the present invention provides a method wherein a test sample is obtained from a subject and GAVE18 protein or nucleic acid (eg mRNA or genomic DNA) is detected. The presence of GAVE18 protein or nucleic acid can be used to diagnose a subject having or at risk of having a disease or condition associated with aberrant GAVE18 expression or activity. A "test sample" as used herein refers to a biological sample from a subject of interest. For example, a test sample can be a biological fluid (eg, serum), a cell sample, or tissue.

In addition, the prognostic assays described herein can be used to determine whether an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) can be administered to a subject to treat patients with abnormal GAVE18 Diseases or conditions associated with expression or activity. For example, these methods can be used to determine whether a particular agent or class of agents (eg, a class of agents that reduce GAVE18 activity) is effective in treating a subject. Accordingly, the present invention provides a method whereby it can be determined whether an agent is effective in treating a patient's condition associated with abnormal GAVE18 expression or activity, wherein a test sample is obtained and GAVE18 protein or nucleic acid is detected (for example, wherein GAVE18 protein or the presence of the nucleic acid can be used to diagnose whether a subject can be treated by administering an agent for a disorder associated with abnormal GAVE18 expression or activity).

The methods of the invention can also be used to detect genetic damage or mutations in the GAVE18 gene, thereby determining whether a subject with a damaged gene is at risk of developing a disorder characterized by abnormal cell proliferation and/or differentiation. In a preferred embodiment, the method comprises detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by the presence of at least one alteration affecting the integrity of the gene encoding the GAVE18 protein Or changes that cause misexpression of the GAVE18 gene. For example, these genetic lesions or mutations can be detected by determining the presence of at least one of the following:

1) One or more nucleotide deletions in the GAVE18 gene;

2) an increase of one or more nucleotides in the GAVE18 gene;

3) One or more nucleotide substitutions in the GAVE18 gene;

4) Chromosomal rearrangements involving the GAVE18 gene;

5) Changes in the messenger RNA transcript level of the GAVE18 gene;

6) Abnormal modification of the GAVE18 gene, such as abnormal changes in the methylation pattern of genomic DNA;

7) the non-wild type level of GAVE18 protein;

8) Allelic loss of the GAVE18 gene; and

9) Inappropriate post-translational modification of GAVE18 protein.

As described herein, there are a number of experimental techniques known in the art that can be used to detect damage in the GAVE18 gene. A preferred biological sample is a sample of peripheral blood leukocytes isolated from a subject by conventional methods.

In certain embodiments, detection of damage involves detection of damage in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Chain reaction (LCR) (see, e.g., Landegran et al., Science (1988) 241:1077-1080; and Nakazawa et al., Proc Natl Acad Sci USA (1994) 91:360-364 ), the latter being particularly useful in detecting point mutations in the GAVE18 gene (see, eg, Abravaya et al., Nucleic Acids Res (1995) 23:675-682). The method may comprise the steps of: collecting a cell sample from a patient, isolating nucleic acid (e.g., genome, mRNA, or both) of the sampled cells, and contacting the nucleic acid sample with one or more primers that specifically hybridize to the GAVE18 gene under certain conditions To achieve hybridization and amplification of the GAVE18 gene (if present), and then detect the presence or absence of the amplified product or detect the size of the amplified product and compare the length with a control sample. It is contemplated that it may be advantageous to use PCR and/or LCR as an initial amplification step in combination with any of the techniques described herein for detecting mutations.

Alternative amplification methods include: self-sustaining sequence amplification (Guatelli et al., Proc Natl Acad Sci USA (1990) 87:1874-1878), transcription amplification system (Kwoh et al., U.S. National Proceedings of the Academy of Sciences (Proc Natl Acad Sci USA) (1989) 86: 1173-1177), Q-β replicase (Lizardi et al., Bio/Technology (1988) 6: 1197) or any other nucleic acid amplification method, which can be used afterwards The amplified molecules are detected by techniques known to those skilled in the art. These detection schemes are especially useful for detecting nucleic acid molecules present in very low quantities.

In an alternative embodiment, mutations in the GAVE18 gene in sample cells can be identified by changes in restriction enzyme cleavage patterns. For example, sample and control DNA can be isolated, amplified (optionally), digested with one or more restriction enzymes, and the length sizes of the fragments determined and compared by gel electrophoresis. A difference in fragment length between sample and control DNA indicates a mutation in the sample DNA. In addition, sequence-specific ribozymes (see, eg, US Pat. No. 5,498,531) can also be used to assess the presence of specific mutations by the creation or loss of ribozyme cleavage sites.

In other embodiments, sample and control nucleic acids (eg, DNA or RNA) can be combined with high-density arrays containing hundreds or thousands of oligonucleotide probes (Cronin et al., HumanMutation (1996) 7:244-255; Kozal et al., Nature Medicine (1996) 2: 753-759) to identify genetic mutations in GAVE18 by hybridization. For example, genetic mutations in GAVE18 can be identified using two-dimensional arrays containing luminescent DNA probes, see, eg, Cronin et al., supra. In short, the first hybridization probe array can be used to scan long-strand DNA in samples and controls, and identify base changes between the two sequences by generating sequentially overlapping probe linear arrays. This step allows the identification of point mutations. This step is followed by a second hybridization array, which allows the characterization of specific mutations by using an array of smaller specific probes that are complementary to all detected variants or mutants. Each mutation array consists of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any sequencing reaction known in the art can be used to directly sequence the GAVE18 gene and detect mutations by comparing the sample GAVE18 sequence with the corresponding wild-type (control) sequence. Examples of sequencing reactions include sequencing reactions as described by Maxam and Gilbert (Proc Natl Acad Sci USA (1977) 74:560) or Sanger (Proc Natl Acad Sci USA (1977) 74:5463 ) developed techniques based on those responses. It is also contemplated to use any of the automated sequencing methods to perform diagnostic tests (Bio/Techniques (1995) 19:448), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al., Adv Chromatogr (1996 ) 36: 127-162; and Griffin et al., Appl Biochem Biotechnol (1993) 38: 147-159).

Other methods that can be used to detect mutations in the GAVE18 gene include methods in which base mismatches in RNA/RNA or RNA/DNA heteroduplexes are detected by protection from cleavage by cleavage agents (Myers et al., Science (1985) 230:1242). In general, "mismatch cleavage" techniques must provide heteroduplexes formed by the hybridization of (labeled) RNA or DNA containing the wild-type GAVE18 sequence to potentially mutated RNA or DNA obtained from a tissue sample. This duplex is treated with a reagent that cleaves single-stranded regions of the duplex that may exist due to, for example, base pair mismatches between the control and sample strands. RNA/DNA duplexes can be treated with RNAase to digest mismatched regions, and DNA/DNA hybrids can be treated with S1 nuclease to digest mismatched regions. In other embodiments, DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmic acid and piperidine to digest mismatched regions. After digestion of the mismatched regions, the resulting material was size-separated on a denaturing polyacrylamide gel to determine the site of the mutation, see e.g. Cotton et al., Proc Natl Acad Sci USA (1988) 85: 4397; Saleeba et al., Methods Enzymol (1992) 217:286-295. In preferred embodiments, control DNA or RNA can be labeled to facilitate detection.

In another embodiment, the mismatch cleavage reaction uses one or more proteins capable of recognizing mismatched base pairs in double-stranded DNA (so-called "DNA mismatch repair enzymes") to detect in a defined system the Point mutations in GAVE18 cDNA and mapped. For example, mutY from E. coli cleaves A at G/A mismatches, and thymidine DNA glycosylase from Hela cells cleaves T at G/T mismatches (Hsu et al., Carcinogenesis (1994) 15:1657-1662 ). According to an exemplary embodiment, a probe based on a GAVE18 sequence (eg, a wild-type GAVE18 sequence) hybridizes to cDNA or other DNA product from a test cell. The double strands are treated with a DNA mismatch repair enzyme and the cleavage products, if any, can be detected in an electrophoretic protocol or similar, see, eg, US Patent No. 5,459,039.

In other embodiments, changes in electrophoretic mobility can be used to identify mutations in the GAvE18 gene. For example, single-strand conformation polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al., Proc Natl Acad SciUSA (1989) 86:2766; see also Cotton, Mutat Res (1993) 285:125-144; Hayashi, Genet Anal Tech Appl (1992) 9:73-79). Single-stranded DNA fragments of sample and control GAVE18 nucleic acids were denatured and then renatured. The secondary structure of single-stranded nucleic acids varies with sequence, resulting in a change in electrophoretic mobility that allows detection of even a single base change. DNA fragments can be labeled or detected with labeled probes. The use of RNA (as opposed to DNA) can enhance the sensitivity of the assay because RNA secondary structure is more sensitive to sequence changes. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate heteroduplex molecules based on changes in electrophoretic mobility. (Keen et al., Trends Genet (1991) 7:5)

In another embodiment, denaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature (1985) 313:495) is used to analyze mutant or wild-type fragments in polyacrylamide gels containing gradient denaturants. in motion. When DGGE is used as an analysis method, the DNA will be modified to ensure that it will not be completely denatured, for example, about 40 bp of high melting point GC-rich DNA, ie, GC clamp, is added by PCR. In another embodiment, a temperature gradient is used instead of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum et al., Biophys Chem (1987) 265:12753).

Examples of other techniques that can be used to detect point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification or selective primer extension. For example, oligonucleotide primers can be prepared in which a known mutation is placed at the center and then hybridized to the target DNA under conditions such that only a perfect match occurs (Saiki et al., Nature (1986) 324:163; Saiki et al., Proc Natl Acad Sci USA (1989) 86:6230). In hybridizing these allele-specific oligonucleotides to PCR amplified target DNA or a number of different mutants, the oligonucleotides can be bound to a hybridization membrane and then hybridized to labeled target DNA.

Alternatively, allele-specific amplification techniques that rely on selective PCR amplification can be used in the present invention. Oligonucleotides used as primers in specific amplification can be in the center of the molecule (so that amplification relies on differential hybridization) (Gibbs et al., Nucleic Acids Res (1989) 17:2437-2448) or in a primer (Prossner, Tibtech (1993) 11:238) carries an interesting mutation. Additionally, it may also be desirable to introduce new restriction sites into the mutated region to create cleavage-based assays (Gasparini et al., Mol Cell Probes (1992) 6:1). It is contemplated that in certain embodiments amplification can also be performed using Taq ligase (Barany, Proc Natl Acad Sci USA (1991) 88:189). In this case, the ligation reaction can only occur if there is a perfect match at the 3' end of the 5' sequence, which allows detection of the presence of known mutations at specific loci by looking for the presence or absence of amplicons .

The methods described herein can be performed, for example, using prepackaged diagnostic kits containing at least one probe nucleic acid or antibody reagent described herein. The method and kit can be conveniently applied, for example, in clinical conditions to diagnose patients showing symptoms of GAVE18 gene-related diseases or disorders or patients with family history of the diseases.

Additionally, any cell type and tissue expressing GAVE18 can be used in the prognostic assays described herein.

3. Pharmacogenomics

Agents or modulators identified by the screening assays described herein that have a stimulatory or inhibitory effect on GAVE18 activity (e.g., GAVE18 gene expression) can be administered to an individual for the treatment (prophylactically or therapeutically) of diseases associated with GAVE18 activity (e.g., Inflammation associated with asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis). It is conceivable to combine this treatment with individual pharmacogenomics, the study of the relationship between an individual's genotype and the individual's responsiveness to foreign compounds or drugs. Differences in the metabolism of therapeutics can lead to severe toxicity or treatment failure by altering the relationship between blood concentration and dose of a pharmacologically active drug. Thus, pharmacogenomics of an individual allows selection of effective prophylactic or therapeutic agents (eg, drugs) based on consideration of the individual's genotype. This pharmacogenomics can also be used to determine appropriate dosage and treatment regimens. Therefore, by determining the activity of the GAVE18 protein, the expression of the GAVE18 nucleic acid, or the mutation content of the GAVE18 gene in the individual, an appropriate therapeutic or preventive agent for the individual can be selected.

Pharmacogenomics deals with clinically significant genetic differences in patient response to drugs resulting from differential and aberrant effects of drug disposition in the patient's body. See, eg, Linder, Clin Chem (1997) 43(2):254-266. In general, two classes of pharmacogenetic conditions can be distinguished. A genetic condition passed on as a single factor that can alter the way a drug acts on the body is called "altered drug action." A genetic condition passed on as a single factor that alters the way the body responds to a drug is called "altered drug metabolism." These pharmacogenetic conditions can exist as rare defects or polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic enzymatic disorder in which the major clinical complications are after ingestion of oxidant drugs (antimalarials, sulfonamides, analgesics, or nitrofurans) and after consumption of fava beans Hemolysis occurs.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of the magnitude and duration of drug action. The discovery of genetic polymorphisms in drug-metabolizing enzymes, such as N-acetyltransferase 2 (NAT2) and the cytochrome P450 enzymes, CYP2D6 and CYP2C19, explains why some patients do not receive their intended drugs after taking standard and safe drug doses effects or exhibit excessive drug response and severe toxicity. These polymorphisms manifest in two phenotypes in the population, the extensive metabolizer (EM) and the poor metabolizer (PM). The prevalence of PM varies between groups. For example, the gene encoding CYP2D6 is highly polymorphic, and several mutations have been identified in PM, all resulting in a lack of CYP2D6 function. Poor metabolizers of CYP2D6 and CYP2C19 very frequently experience excessive drug reactions and side effects after receiving standard doses. As evidenced by the analgesic effects of codeine (mediated by the metabolite morphine formed by CYP2D6), PM would not exhibit a therapeutic response if the metabolite was the active therapeutic moiety. At the other extreme are so-called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultrarapid metabolism has been identified, which results from CYP2D6 gene amplification.

Therefore, by determining the activity of GAVE18 protein, the expression of GAVE18 nucleic acid, or the mutation content of GAVE18 gene in an individual, an appropriate agent for prevention or treatment of an individual can be selected. In addition, pharmacogenomic studies can be used to identify drug response phenotypes in individuals by genotyping polymorphic alleles encoding drug-metabolizing enzymes. When treating a subject with a modulator of GAVE18 (e.g., a modulator identified by one of the exemplary screening assays described herein), this information may allow for the avoidance of adverse effects or treatment failure in dosing or selection of drugs, thereby enhancing treatment or preventive efficacy.

4. Monitoring efficacy during clinical trials

Monitoring the effect of agents (eg, drugs or compounds) on the expression or activity of GAVE18 (eg, the ability to regulate abnormal cell proliferation and/or differentiation) can be applied in both basic drug screening and clinical trials. For example, the effectiveness of agents (as determined by the screening assays described herein) in increasing GAVE18 gene expression, protein levels, or protein activity can be monitored in clinical trials of subjects exhibiting reduced GAVE18 gene expression, protein levels, or protein activity. sex. Alternatively, the effectiveness of agents (as determined by the screening assays described herein) in reducing GAVE18 gene expression, protein levels, or protein activity can be monitored in clinical trials in subjects exhibiting increased GAVE18 gene expression, protein levels, or protein activity. sex. In these clinical trials, the expression or activity of GAVE18 and, preferably, the expression or activity of other genes (eg, genes involved in cell proliferative diseases) can be used as markers of the immune responsiveness of specific cells. For example, without limitation, genes (including GAVE18) that can be regulated in cells when treated with agents (e.g., compounds, drugs, or small molecules) that modulate GAVE18 activity (e.g., agents identified by the screening assays described herein) can be identified . Thus, for example, in a clinical trial, to study the effect of an agent on a cell proliferative disease, cells can be isolated, RNA prepared and analyzed for the expression levels of GAVE18 and other genes involved in the disease. The level of gene expression (i.e., gene expression-like) can be quantified by performing Northern blot analysis or RT-PCR as described herein, or, alternatively, measuring the amount of protein produced using one of the methods described herein or The activity level of GAVE18 or other genes is measured. In this way, gene expression can serve as a marker indicative of a cell's physiological response to an agent. Thus, response status can be determined prior to and at various points during treatment of an individual with an agent.

In a preferred embodiment, the invention provides a method whereby the use of agents (e.g., agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids, small molecule or other drug candidates) to treat the efficacy of the subject, the method comprises the steps of: (i) obtaining a pre-drug sample from the subject before administering the medicament; (ii) detecting the level of GAVE18 protein, mRNA or genomic DNA in the pre-drug sample expression level; (iii) obtain one or more samples after administration from the subject; (iv) detect the expression or activity level of GAVE18 protein, mRNA or genomic DNA in the sample after administration; (v) analyze the GAVE18 protein in the sample before administration comparing the expression or activity level of GAVE18 protein, mRNA or genomic DNA with the expression or activity level of GAVE18 protein, mRNA or genomic DNA in one or more post-dose samples; and (vi) altering the patient's drug administration regimen accordingly. For example, it may be desirable to increase the administration of the agent to increase the expression or activity level of GAVE18 beyond the level detected, ie, to increase the efficacy of the agent. Alternatively, it may be desirable to reduce the administration of the agent to reduce the expression or activity level of GAVE18 below the level detected, ie, to reduce the efficacy of the agent.

D. Treatment

The present invention provides methods of prevention and treatment for patients at risk of (or susceptible to) or already suffering from diseases associated with aberrant GAVE18 expression or activity. Such diseases include, but are not limited to, eg, inflammatory diseases such as asthma, chronic obstructive pulmonary disease and rheumatoid arthritis.

1. Prevention methods

In one aspect, the invention provides methods of preventing a disease or condition associated with aberrant GAVE18 expression or activity in a subject by administering to the subject an agent that modulates GAVE18 expression or at least one GAVE18 activity. Whether an individual is at risk for a disease that may be caused or contributed to by aberrant GAVE18 expression or activity can be identified, for example, by any one or combination of diagnostic or prognostic tests described herein. The prophylactic agent can be administered before symptoms characterized by GAVE18 abnormality develop, in order to prevent the disease or disorder, or alternatively arrest the progression of the disease or disorder. For example, depending on the type of GAVE18 abnormality, the individual can be treated with a GAVE18 agonist or a GAVE18 antagonist. Suitable agents can be determined according to the screening assays described herein.

2. Treatment

Another aspect of the invention relates to methods of modulating the expression or activity of GAVE18 for therapeutic purposes. The modulating methods of the invention involve contacting a cell with an agent capable of modulating the activity of one or more GAVE18 proteins associated with the cell. Agents capable of modulating the activity of GAVE18 protein can be agents described herein, such as nucleic acids or proteins, naturally associated ligands of GAVE18 protein, peptides, GAVE18 peptidomimetics, or other small molecules. In one embodiment, the agent stimulates one or more biological activities of the GAVE18 protein. Examples of such stimulators include active GAVE18 protein and nucleic acid molecules encoding GAVE18 that have been introduced into cells. In another embodiment, the agent inhibits one or more biological activities of the GAVE18 protein. Examples of such inhibitors include antisense GAVE18 nucleic acid molecules and anti-GAVE18 antibodies. This modulation method can be performed in vitro (eg, by culturing the cells with the agent) or, alternatively, in vivo (eg, by administering the agent to a patient). Thus, the present invention provides methods of treating an individual suffering from a disease or disorder characterized by aberrant expression or activity of a GAVE18 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (eg, an agent identified by a screening assay described herein) or combination of agents that modulates (eg, up-regulates or down-regulates) GAVE18 expression or activity. In another embodiment, the method involves administering a GAVE18 protein or nucleic acid molecule as a therapy to compensate for decreased or aberrant GAVE18 expression or activity.

Stimulation of GAVE18 activity is advantageous when GAVE18 is aberrantly downregulated and/or when increased GAVE18 activity may have beneficial effects. Conversely, inhibition of GAVE18 activity is advantageous when GAVE18 is aberrantly upregulated and/or when reduced GAVE18 activity may have beneficial effects.

The present invention may be better understood by reference to the following non-limiting examples, which are provided as illustrations of the invention. The following examples are given to more fully illustrate the preferred embodiments of the invention. However, it is not intended to be construed as limiting the scope of the invention.

Example

Identification of GAVE18: The FASTA algorithm (Wisconsin GCG software package, version 10.1) was used to perform a homology search on the human genome sequence database HTG (NCBI/NIH) by querying multiple GPCRs. The returned statistically significant homologous genomic DNA sequences were translated into three forward frames for BLASTp searches against protein databases. The genomic DNA sequence AC083865 from chromosome 7 was determined to contain a putative GPCR sequence, which was subsequently designated GAVE18. The chromosomal location of GAVE18 was mapped to p14.1.

Cloning of genomic DNA encoding GAVE18: Primers specific to the predicted 5' and 3' sequences of GAVE18 were designed. Forward primer HP271, AAA ACT GCA TGC TGT GGCTGC (SEQ ID NO: 3), and reverse primer HP272, TTT CAG CTG AGC CCAGAA CTC (SEQ ID NO: 4), for amplification by polymerase chain reaction (PCR) For amplification of GAVE18 genomic DNA, human genomic DNA was used as a template. The PCR conditions were as follows: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. The above process was performed for 35 cycles, and then extended at 72°C for 5 minutes. The amplified DNA fragment was cloned into the pCRII-TOPO vector from Invitrogen. The DNA insert of this clone was confirmed by DNA sequencing. All PCR amplifications were performed in a DNA Engine Tetrad (MJ Research, model PTC-225).

Northern blot analysis: Human multiple tissue Northern blot material from Clontech was hybridized to a full-length open reading frame DNA fragment labeled with [α- ^32P ]dCTP according to the manufacturer's instructions. The blotted material after hybridization was washed with 2XSSPE and 0.1% SDS at 50°C for 30 minutes, and with 0.1XSSPE and 0.1% SDS at 50°C for 1 hour. The blotted material was then exposed to X-ray film at -70°C in the presence of an intensifying screen.

Taqman analysis: Total RNA from human tissue was purchased from Clontech. Prior to cDNA generation, total RNA was treated with DNAseI to avoid potential genomic DNA contamination. Briefly, total RNA was mixed with 5 μl 10x DNAseI buffer (20 mM Hepes pH 7.5; 10 mM CaCl ₂ ; 10 mM MgCl ₂ ; 1 mM DTT and 50% (v/v) glycerol) (Ambion), RNAse inhibitor and 1 μl RNase-free DNAseI (2 U/μl; Ambion) was mixed in a final volume of 50 μl at 37° C. for 1 hour. After the phenol precipitation step, cDNA synthesis was performed using the Superscript selection system as described by Life Technologies. Taqman primers/probes were designed with Primer Express 1.0 software (ABI). The GAVE18 amplicon spanned 72 nucleotides of the open reading frame, with a forward primer: 5'GATTCTGTTGGTCTTCCAGGTCTT 3' (SEQ ID NO: 5), and a reverse primer: 5'CCAGAACTCCTGGTGGGATAGT 3' (SEQ ID NO: 6 ), and the Taqman probe sequence is: 5'FAM-TGGCGTAGCTTCTGCACCATCAACA-TAMRA 3' (SEQ ID NO: 7). Fam was used as reporter dye and Tamra as quencher. Taqman probes were synthesized by Operon Technologies upon request. Taqman reactions were carried out in 96-well plate MicroAmp optical tubes (PE), with a final volume of 50 μl, containing: 25 μl Taqman PCR mix (Perkin Elmer); 1 μl forward primer with a final concentration of 900 nM; 1 μl reverse primer with a final concentration of 900 nM; and 1 μl Taqman probe at a final concentration of 200 nM; 5 μl cDNA template (calculated concentration of 10 ng/μl) and 17 μl water. Taqman PCR conditions were performed as described in PE Applied Biosystem. Human β-actin primer probe (designed and purchased from PE applied Biosystem) was used as an internal control. Duplicate Taqman reactions were performed for each tissue, target gene and internal control. In addition, a standard curve was drawn twice against human β-actin using increasing amounts of total brain cDNA. This allows us to obtain the relative number of amplicons that were amplified. Expression of target genes is expressed as relative fold expression relative to brain cDNA.

PCR screening of cDNA library: specific PCR primers for GAVE18 coding region: 5' AAA ACT GCA TGC TGT GGC TGC 3' (SEQ ID NO: 8), and 5' TTT CAGCTG AGC CCA GAA CTC 3' (SEQ ID NO: 9) For screening the combined spleen, placenta and kidney cDNA libraries. PCR screening was performed in a 96-well plate using the following PCR method: 94°C for 3 minutes; then 40 cycles of 94°C for 30 seconds, 52°C for 30 seconds, and 68°C for 45 seconds. Positive subpools were then diluted for the next round of PCR screening. A limited number of colonies from positive subpools were plated on agar plates and positive plasmids were confirmed by PCR followed by DNA sequencing analysis.

Description of results

GAVE18 has a restricted expression pattern in the immune system, mainly expressed in bone marrow, peripheral blood leukocytes, spleen and thymus. Inhibition or activation of the GAVE18 GPCR receptor modulates immune cell maturation, development, and responses during inflammation. GAVE18 is upregulated by the cytokine TNF-α in granulocytes. TNF-α plays an important role in many inflammatory diseases. GAVE18 is also induced by TNF-α in bronchial epithelial cells, and GAVE18 is an excellent drug target for respiratory diseases such as asthma. GAVE18 is also a good target for other inflammatory diseases such as RA and COPD.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, many modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such changes are also within the scope of the appended claims.

It is also to be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weight values, are approximate and are provided for the purpose of description.

Several publications are cited herein, the disclosures of which are hereby incorporated by reference in their entirety.

Claims (29)

1. An isolated nucleic acid molecule comprising the DNA sequence (SEQ ID NO: 1) of Figure 5. 2. An isolated nucleic acid molecule hybridizable to the isolated nucleic acid molecule of claim 1 under stringent hybridization conditions, or a hybridization probe complementary to the isolated nucleic acid molecule of claim 1 under stringent hybridization conditions. 3. The isolated nucleic acid molecule of claim 1 or 2, which encodes a polypeptide having the amino acid sequence of Figure 6 (SEQ ID NO: 2). 4. The isolated nucleic acid molecule of claim 2, which encodes a polypeptide having an amino acid sequence at least 30% identical to the amino acid sequence of SEQ ID NO:2. 5. The isolated nucleic acid molecule of claim 1 or 2, which is detectably labeled. 6. The detectably labeled isolated nucleic acid molecule of claim 5, wherein said detectable label comprises an enzyme, a radioisotope, or a fluorescent chemical. 7. An isolated polypeptide comprising the amino acid sequence of Figure 6 (SEQ ID NO: 2). 8. An isolated nucleic acid molecule encoding the purified polypeptide of claim 7. 9. The purified polypeptide of claim 7, which is detectably labeled. 10. The purified polypeptide of claim 9, wherein the detectable label comprises an enzyme, a radioisotope, or a fluorescent chemical. 11. An antibody produced using the purified polypeptide of claim 7 as an immunogen. 12. The antibody of claim 11, wherein the antibody is selected from a monoclonal antibody, a polyclonal antibody or a chimeric antibody. 13. The antibody of claim 11 which is detectably labeled. 14. The antibody of claim 13, wherein the detectable label comprises an enzyme, a radioisotope, or a fluorescent chemical. 15. An expression vector comprising the isolated nucleic acid molecule of claim 1 operably linked to an expression control element. 16. An expression vector comprising the isolated nucleic acid molecule of claim 2 operably linked to an expression control element. 17. The expression vector of claim 15 or 16, wherein the expression regulatory elements are selected from the group consisting of constitutive regulatory sequences, cell-specific regulatory sequences and inducible regulatory sequences. 18. The expression vector of claim 17, wherein the expression control element is a promoter. 19. The expression vector of claim 18, wherein the promoter comprises the immediate early promoter of hCMV, the early promoter of SV40, the early promoter of adenovirus, the early promoter of vaccinia virus, the early promoter of polyoma virus, Late promoter of SV40, late promoter of adenovirus, late promoter of vaccinia virus, late promoter of polyomavirus, lac system, trp system, TAC system, TRC system, main operator gene and promoter region of lambda phage , the regulatory region of the fd coat protein, the 3-phosphoglycerol kinase promoter, the acid phosphatase promoter or the yeast alpha mating factor promoter. 20. A host cell transformed or transfected with the expression vector of claim 15 or 16. 21. The host cell of claim 20, wherein said host cell comprises a prokaryotic cell or a eukaryotic cell. 22. The host cell of claim 21, wherein said host comprises E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1 .1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells. 23. A method for producing the isolated polypeptide of claim 7, comprising the steps of: a) culturing the host cell of claim 20 under conditions that provide expression of said isolated polypeptide; and b) recovering said isolated polypeptide from said host, said culture or a combination thereof. 24. A method of treatment for modulating GAVE18 signaling activity or signal transduction in a patient in need thereof, said method of treatment comprising administering to said patient an agonist, antagonist or inverse agonist of GAVE18. 25. A method for identifying an agonist of GAVE18, comprising: contacting a potential agonist with a cell expressing GAVE18, and determining whether the signaling activity of GAVE18 in the presence of the potential agonist is relative to the absence of the potential agonist The activity of GAVE18 increased during the time. 26. A method for identifying an inverse agonist of GAVE18, comprising: contacting a potential inverse agonist with a cell expressing GAVE18, and determining whether the activity of GAVE18 is relative to that in the presence of said potential inverse agonist The activity of GAVE18 is reduced in the absence of such potential inverse agonists, and is also reduced in the presence of endogenous ligands or agonists. 27. A method for identifying an antagonist of GAVE18 comprising: contacting a potential antagonist with a cell expressing GAVE18, and determining whether the signaling activity of GAVE18 in the presence of said potential antagonist is relative to the presence of endogenous ligand The activity of GAVE18 is reduced in the presence of agonists or agonists. 28. A therapeutic composition comprising an agonist, antagonist or inverse agonist of GAVE18 capable of modulating the signaling activity or transduction of GAVE18. 29. A method of treating a disease comprising administering to a patient in need thereof a therapeutic composition comprising an agonist, antagonist or inverse agonist of GAVE18 capable of modulating the signaling activity or transduction of GAVE18.

Images