JP2005514934A - Nucleic acids encoding G protein-coupled receptors and uses thereof - Google Patents

Abstract

  Provided here are novel and useful G protein-coupled receptors involved in signal transduction associated with inflammation and physiological immune responses. Also provided are methods for screening for molecules that can modulate the activity of the receptor using the receptor. Such molecules are readily applicable in treating excess inflammation and diseases and disorders related to immunity.

Description

  The present invention generally relates to a novel nucleic acid molecule encoding a previously unknown G protein-coupled receptor, GAVE6, and the use of the nucleic acid molecule and GAVE6.

  G protein-coupled receptors (GPCRs) are a large family of transmembrane membrane proteins that are involved in cell signaling. GPCRs can respond to and transduce a wide variety of extracellular signals, including neurotransmitters, hormones, odorants and light stimuli, and initiate intracellular second messenger responses. Many therapeutic agents target GPCRs because these receptors mediate highly diverse physiological responses such as inflammation, vasodilation, heart rate, tracheal dilatation, endocrine and peristalsis Because.

GPCRs are characterized by an extracellular domain, seven transmembrane domains and an intracellular domain. Some of the functions performed by receptors, such as binding to ligands and interaction with G proteins, are related to the presence of specific amino acids at critical sites. For example, various studies show that differences in the amino acid sequence of GPCRs explain differences in affinity with natural ligands or small molecule agonists or antagonists. In other words, slight differences in sequence can cause differences in different binding affinities and activities (eg Meng et al., J Bio Chem (1996) 271 (50): 32016- 20; Burd et al., J Bio Chem (1998) 273 (51): 34488-95; and Hurley et al., J Neurochem (1999).
72 (No. 1): see pages 413-21). In particular, research has revealed that differences in the amino acid sequence of the intracellular third domain result in different activities. Myburgh et al. Found that 261st alanine in the third intracellular loop of the gonadotropin-releasing hormone receptor is essential for G protein coupling and intracellular uptake of the receptor (Biochem J (1998) 331 ( Part 3): 893-6). Wonerow et al. Studied the thyrotropin receptor and demonstrated that deletion in the third intracellular loop results in constitutive receptor activity (J Bio Chem (1998) 273 ( 14): 7900-5).

In general, binding of an endogenous ligand to a receptor results in a structural change in the intracellular domain of the receptor, which allows conjugation of the intracellular domain to the G protein of the intracellular component. There are many types of G proteins, for example G _q, G _s, G _i, G _z and G _o are mentioned (e.g., Dessauer, etc., Clin Sci (Colch) (1996 years) Volume 91 (No. 5): 527-37). The IC-3 loop and 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., Supra). Some GPCRs are “promiscuous” with respect to G proteins, ie, GPCRs can interact with more than one G protein (eg, Kenakin, Life Sciences (1988) 43: 1095 Page).

  The ligand-activated GPCR couples with the G protein and initiates a signal transduction cascade process (called “signal transduction”). Such signaling ultimately results in cell activation or cell suppression.

GPCRs exist in the cell membrane in an equilibrium between two different conformations, an “inactive” and an “active” state. Inactive receptors cannot be linked to intracellular signaling pathways to trigger biological responses (exceptions such as receptor overexpression in transformed cells exist, eg www.creighton.edu/ See Pharmacology / inverse.htm ). By adjusting the conformation to the active state, it can be linked to the transmission pathway (via the G protein) and a biological reaction occurs. By binding an agonist, the active three-dimensional structure is made easier. However, sometimes such receptors are said to be constitutively active if there is already a substantial response without any agonist at all (ie already in an active conformation, or , In a ligand-independent or autonomous active state). When agonists are added to such systems, a more enhanced response is usually observed. However, when conventional antagonists are added, the binding of such molecules does not produce any effect. On the other hand, certain antagonists cause inhibition of receptor constitutive activity, suggesting that this latter class of drugs is technically not an antagonist but an agonist with negative intrinsic activity. . Such drugs are called inverse agonists. ( Www.creighton.edu/Pharmacology/inverse.htm )

  The hypothesis that previous research on receptors was the starting point was to identify endogenous ligands first, and then identify molecules that act on antagonists and other receptors prior to their discovery. Even when antagonists were first discovered, a dogmatic approach was to identify endogenous ligands (WO00 / 22131). However, since the active state is most useful for assay screening purposes, in the absence of information on endogenous ligands, obtaining such constitutive receptors, particularly GPCRs, allows agonists, partial agonists Allows easy isolation of inverse agonists and antagonists. In addition, drugs that cause inhibition of constitutive activity in diseases caused by impaired receptor activity, or more specifically reduce the concentration of effective activated receptors, are receptors in an autonomously active state. It can be easily found by assay using. For example, for receptors that can be transfected into a patient to treat a disease, the inverse agonist discovered by the above-described assay allows for subtle modulation of the activity of such receptors.

  Diseases such as asthma, chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis (RA) are generally due to inflammatory pathogenesis caused by helper T cells, monocytes-macrophages and eosinophils. Current anti-inflammatory treatments with corticosteroids are effective for asthma but are associated with metabolic and endocrine side effects. The same is probably true for inhalation formulations that are absorbed through the lung or nasal mucosa. There are currently no satisfactory oral treatments for RA or COPD.

  Eosinophils mediate many of the airway dysfunctions in allergies and asthma. Interleukin-5 (IL-5) is an eosinophil proliferation and activated cytokine. Studies have shown that IL-5 is required for tissue eosinophilia and eosinophil-mediated tissue damage resulting in airway hyperreactivity (Chang et al., J Allergy Clin Immunol (1996) 98 (5 pt 1): 922-931 and Duez et al., Am J Respir Crit Care Med (2000) 161 (1): 200-206). IL-5 is produced by helper T2 cells (Th2) after exposure to allergens (eg, house dust mite antigen) in atopic asthma.

  RA is believed to result from the accumulation of activated macrophages in the damaged synovium. Interferon-γ (IFN-γ) is a cytokine derived from helper T1 cells (Th1) and has a property of promoting many inflammations. This is the most powerful macrophage activating cytokine and induces MHC class II gene transcription resulting in the formation of a dendritic cell-like phenotype.

  Lipopolysaccharide (LPS) is a component of the Gram-negative bacterial cell wall and causes an inflammatory response, including the release of tumor necrosis factor alpha (TNFα). In RA, the effect of treatment with intravenous administration of anti-TNFα antibody has been clinically demonstrated. COPD is also thought to result from the accumulation of macrophages in the lung, which generate neutrophil chemoattractants (eg, IL-8: de Boer et al., J Pathol (2000) 190 (5). ): 619-626). Both macrophages and neutrophils release cathepsins that lead to alveolar wall collapse. It is believed that lung epithelial tissue can be an important source of inflammatory cell chemoattractants and other inflammatory cell activators (eg, Thomas et al., J Virol (2000) 74 (18 ): 8425-8433; Lamkhioued et al., Am J Respir Crit Care Med (2000) 162 (2 Pt.1): 723-732; and Sekiya et al., J Immunol (2000) 165 (4) ): See pages 2205-2213).

  Given the role played by GPCRs in disease and the ability to treat diseases by modulating GPCR activity, the identification and characterization of previously unknown GPCRs has led to novel novel treatments for conditions associated with GPCR activity. Development of compositions and methods can result. Therefore, what is needed is the discovery, isolation, and characterization of new and useful nucleic acid molecules that encode previously unknown GPCRs.

  What is further needed is an assay that utilizes such previously unknown GPCRs to identify potential agonist or antagonist molecules for a particular GPCR. Such molecules can be readily applied as therapeutic agents that modulate GPCR activity in the body and, as a result, can treat many diseases associated with GPCR activity.

  Citation of any reference herein shall not be construed as an admission that such reference is available as “Prior Art” to the instant application.

  The present invention provides methods and compositions that identify and characterize a novel and constitutively active GPCR, GAVE6, and apply this discovery to the identification and treatment of related diseases.

  To this end, in a broad sense, the present invention encompasses an isolated nucleic acid molecule comprising the DNA sequence of FIG. 1 (SEQ ID NO: 1), a variant thereof, a fragment thereof, or an analog or derivative thereof. Such variants of the invention may be allelic variants, degenerate variants, allelic variants that result in degenerate conversion of sequences.

  Furthermore, the present invention includes an isolated nucleic acid molecule that can hybridize to the isolated nucleic acid molecule of SEQ ID NO: 1 under stringent hybridization conditions, or a variant thereof. Still further, the present invention includes an isolated nucleic acid molecule capable of hybridizing under stringent hybridization conditions to a nucleic acid molecule complementary to the DNA sequence of SEQ ID NO: 1. Stringent hybridization conditions are described later.

  The invention also encompasses an isolated nucleic acid molecule containing a DNA sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

  If necessary, the above-described isolated nucleic acid molecule of the present invention may be detectably labeled. Examples of detectable labels used herein include, but are not limited to, enzymes, radioisotopes, or fluorescent chemicals. Specific examples of detectable labels are described later.

Certain polypeptides are also encompassed by the present invention. For example, the present invention encompasses a purified polypeptide containing the amino acid sequence of SEQ ID NO: 2, a conservative variant thereof, or an analog or derivative thereof. If necessary, the polypeptides of the invention may be detectably labeled.

  The present invention further includes an antibody using the polypeptide of the present invention as an immunogen for antibody production. Such antibodies can be monoclonal or polyclonal. Furthermore, the antibodies may be “chimeric”, for example, they may contain proteins of the domain of an antibody made in a different animal species against the purified polypeptide of the invention. In certain embodiments, the antibodies of the invention may be “humanized”. Usually, the antibodies of the present invention are detectably labeled. Specific examples of detectable labels applied here will be described later.

  The present invention encompasses an expression vector containing a nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1, a variant thereof, an analog or derivative thereof, or a fragment thereof operably linked to an expression control element. Furthermore, the present invention provides an isolated nucleic acid capable of hybridizing under stringent hybridization conditions to an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1 operatively linked to an expression control element. To a molecule or 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 an expression control element. An expression vector comprising an isolated nucleic acid molecule capable of hybridizing under hybridization conditions. A specific example of an expression control element used herein is a promoter. Examples of specific promoters that can be used in the present invention include, but are not limited to: HCMV early promoter, SV40 early promoter, adenovirus early promoter, vaccinia early promoter, polyoma early promoter, SV40 late promoter, adenovirus late promoter, vaccinia late promoter, polyoma late promoter, lac / trp system, TAC system, TRC system Lambda major operator and promoter region, coat protein regulatory region, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or yeast alpha mating factor promoter.

  Using the expression vector of the present invention, a host cell can be transformed or transduced to produce a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof. The host cell can be a prokaryotic cell or a eukaryotic cell. Specific examples of unicellular hosts used here include E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, RI. I, BW, LM, COS1, COS7, BSC1, BSC40, BMT10, and Sf9 cells.

  Furthermore, the present invention also includes a method for producing a purified polypeptide comprising the amino acid sequence of SEQ ID NO: 2, a variant thereof or a fragment thereof. Such methods involve culturing host cells transformed or transduced with the expression vectors of the invention under conditions that allow expression of the purified polypeptide, and then unicellular host, the culture medium of the host cells, or both. From which the purified polypeptide is recovered.

  Furthermore, the present invention encompasses assays that identify compounds that can modulate GAVE6 activity. Such compounds can be agonists, antagonists or inverse agonists of GAVE6. Therefore, the present invention is a method for identifying an agonist of GAVE6, comprising contacting an agonist candidate substance with a cell expressing GAVE6 in the presence of an endogenous ligand, and GAVE6 when the agonist candidate substance is present. Measuring whether or not the signal activity of the signal increases in comparison with the signal activity of GAVE6 in the absence of the agonist candidate substance.

Similarly, the present invention encompasses methods for identifying inverse agonists of GAVE6. Such a method involves contacting an inverse agonist candidate substance with a cell expressing GAVE6, and the signal activity of GAVE6 when an inverse agonist candidate substance and an endogenous ligand or agonist are present is different from that of the endogenous ligand or agonist. Measuring whether it decreases compared to the signal activity of GAVE6 in the absence of an agonist candidate substance, and whether it decreases in the presence of an endogenous ligand or agonist.

  Of course, the present invention also encompasses a method of identifying a GAVE6 antagonist. In such a method, an antagonist candidate substance is contacted with a cell expressing GAVE6, and the signal activity of GAVE6 in the presence of the antagonist candidate substance is compared with the signal activity of GAVE6 in the presence of an endogenous ligand or agonist. And measuring whether it decreases.

  That is, an object of the present invention is to provide an isolated nucleic acid sequence encoding a GAVE6 protein, fragment thereof or variant thereof.

  Variants of nucleic acid molecules comprising the DNA sequence of SEQ ID NO: 1 or isolated nucleic acid molecules that are capable of hybridizing to SEQ ID NO: 1 under stringent conditions are also an object of the present invention.

  Furthermore, it is an object of the present invention to provide the amino acid sequence of GAVE6, its mutants, its fragments or similar substances or its derivatives.

  Furthermore, it is also an object of the present invention to provide an expression vector that encodes GAVE6, a variant thereof, a fragment thereof, or an analog or derivative thereof, wherein the DNA sequence is operably linked to an expression control element. is there.

  Furthermore, it is an object of the present invention to provide an antibody using GAVE6, a mutant thereof, a similar substance or derivative thereof, or a fragment thereof as an immunogen.

  Still further, another object of the present invention is a method for identifying a compound capable of modulating GAVE6 protein activity. Such a modulator may be an antagonist of GAVE6, an agonist of GAVE6 or an inverse agonist of GAVE6.

  Still further, it is an object of the present invention to provide a pharmaceutical composition for use in modulating the activity of GAVE6. Such regulatory effects can be used to treat various diseases associated with GAVE6 activity, such as various inflammatory diseases, asthma, chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis, to name a few. .

  These and other aspects of the present invention may be better understood with reference to the following drawings and detailed description.

DETAILED DESCRIPTION OF THE INVENTION As explained above, the present invention is a surprising and anticipation of an unknown nucleic acid molecule that encodes a previously unknown G protein-coupled receptor, herein named GAVE6. Related to the discovery. In particular, it has been discovered that GAVE6 is expressed in immune organs or organs such as kidney, liver and small intestine.

In the present specification and claims, various terms and phrases used to describe the present invention are defined as follows:
As used herein, the term “modulator” refers to components that modulate GAVE6 activity, such as, but not limited to, ligands and candidate compounds. The modulator of the present invention may be an agonist, partial agonist, antagonist or inverse agonist of GAVE6.

  As used herein, the term “agonist” refers to a component that activates an intracellular response when bound to a receptor or promotes binding of GTP to a membrane (eg, without limitation, a ligand and Candidate compound).

  The term “partial agonist” as used herein activates intracellular responses to a lesser extent than an agonist when bound to a receptor, or the binding of GTP to a membrane is weaker than an agonist. Refers to components that promote to a degree (eg, but not limited to, ligands and candidate compounds).

  As used herein, the term “antagonist” refers to components that competitively bind to the same site on the receptor as the agonist (eg, but are not limited to, ligands and candidate compounds). However, antagonists do not activate intracellular responses initiated by activated receptors, but can inhibit intracellular responses by agonists or partial agonists. In this regard, antagonists do not reduce the baseline intracellular response in the absence of an agonist or partial agonist.

  As used herein, the term “inverse agonist” refers to components that bind to constitutively activated receptors and inhibit baseline intracellular responses, such as, but not limited to, ligands and candidate compounds. . This baseline intracellular response is initiated by the active form of the receptor and is observed in the absence of an agonist or partial agonist, or below normal basal levels of activity that reduce GTP binding to the membrane.

  As used herein, the term “candidate compound” refers to components (eg, but not limited to ligands and candidate compounds) that are amenable to screening methods. In one embodiment, the term does not include compounds that are publicly known to be compounds selected from the group consisting of agonists, partial agonists, inverse agonists or antagonists of GAVE6. Such compounds include the identification of endogenous ligands specific for the receptor, and / or methods of screening candidate compounds for receptors that require competitive assays to assess efficacy. Identified by the discovery method.

  As used herein, the terms “constitutively activated receptor” or “autonomously activated receptor” are used interchangeably herein and are receptors activated in the absence of a ligand. Means the body. Such constitutively active receptors can be endogenous (eg, GAVE6) or non-endogenous; that is, the GPCR is modified to create a constitutive variant of the wild-type GPCR by recombinant methods. (See, eg, EP1071701; WO 00/22129; WO 00/22131; and US 6,150,393 and US 6,140,509, which are incorporated herein by reference in their entirety).

  As used herein, the term "constitutive receptor activation" means that the receptor is stabilized in an active state by means other than binding of the endogenous ligand or its chemical equivalent to the receptor. To do.

  As used herein, the term “ligand” means a component that binds to another molecule, which component includes, but is in no way limited to, hormones or neurotransmitters, and further, this component is a receptor. It binds stereoselectively to the body.

  As used herein, the term “family” when referring to a protein or nucleic acid molecule of the present invention has a structural domain that is common in appearance and is sufficient in amino acid or nucleic acid sequence identity as defined herein. It is intended to mean two or more proteins or nucleic acid molecules that have sex. Such family members can be naturally occurring and can be from the same or different species. For example, one family consists of a first protein from human and a homologue of that protein from rodent, as well as a second protein from different human and its second protein from rodent Homologues can be included. Family members may also have common functional properties.

  As used herein interchangeably, the terms “GAVE6 activity”, “biological activity of GAVE6” and “functional activity of GAVE6” are caused by a GAVE6 protein, peptide or nucleic acid molecule in GAVE6-responsive cells. Means activity that can be measured in vivo or in vitro by standard techniques. GAVE6 activity may be a direct action such as ligation to a second protein or enzymatic activity on the second protein, or an indirect such as cellular signal activity mediated by the interaction of the second protein with the GAVE6 protein. Active. In certain embodiments, GAVE6 activity includes, but is not limited to, at least one or more of the following activities: (i) an activity capable of interacting with a protein in the GAVE6 signaling pathway; ) An activity capable of interacting with a GAVE6 ligand; (iii) an activity capable of interacting with an intracellular target protein.

  Furthermore, in the context of the present invention, those skilled in the art can employ standard molecular biology, microbiology, and recombinant DNA techniques. Such techniques are explained fully in the literature. References to be referred are, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (hereinafter "Sambrook et al., 1989") DNA Cloning: A Practical Approach, Volumes I and II (DN Glover, 1985); Oligonucleotide Synthesis (MJ Gait, 1984); Nucleic Acid Hybridization (co-edited by BD Hames and SJ Higgins). (1985)]; Transcription And Translation [B. D. Hames and S. J. Higgins co-edited. (1984)]; Animal Cell Culture [R. I. Freshney. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (Co-edition), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

  Accordingly, as used herein, the following terms have the definitions set forth below.

  A “vector” is a replicon. A few examples include plasmids, phages or cosmids, to which other DNA fragments can be ligated so that the fragments bound to them can replicate. A “replicon” is an autonomous unit of DNA replication in vivo, ie, all genetic elements (eg, plasmids, chromosomes, viruses) that function to allow replication under its own control. Specific examples of vectors are described below.

  “Cassette” means a DNA fragment that can be inserted into a vector at a specific restriction enzyme recognition site. This DNA fragment encodes the polypeptide of interest, and the cassette and restriction enzyme recognition sites are designed to ensure that the cassette is inserted in a correct reading frame for transcription and translation.

A cell is “transfected” by exogenous or heterologous DNA when such DNA is introduced into the cell. A cell has been “transformed” by exogenous or heterologous DNA when the transfected DNA causes a phenotypic change. Preferably, the transforming DNA will be integrated (covalently linked) into the chromosome to form the genome of the cell.

  By “heterologous” DNA is meant DNA that is not naturally present in the cell or in a chromosomal site of the cell. Preferably, the heterologous DNA comprises a gene that is foreign to the cell.

  “Homologous recombination” means insertion of a foreign DNA sequence of a vector into a chromosome. In particular, the vector targets a specific site on the chromosome for homologous recombination. For specific homologous recombination, the vector has a region of homology that is sufficiently long with the sequence of the chromosome to allow the vector to complementarily bind and incorporate into the chromosome. The longer the region of homology and the higher the degree of sequence similarity, the greater the efficiency of homologous recombination.

In one aspect, the present invention provides an isolated nucleic acid molecule comprising the DNA sequence of FIG. 1 (SEQ ID NO: 1), a variant, fragment or analog thereof, or derivative thereof. is there.

  “Nucleic acid molecule” is a phosphate polymer of ribonucleoside (adenosine, guanosine, uridine or cytidine; “RNA molecule”) or deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; “DNA molecule”), or , All phosphate ester analogs thereof, such as phosphorothioates or thioesters, which are single-stranded or double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule means only the primary and secondary structure of the molecule, notably in DNA or RNA molecules, and is not limited to any particular tertiary structure. To that end, the term includes double-stranded DNA as found, inter alia, in linear or circular DNA molecules (eg, restriction enzyme fragments), plasmids, and chromosomes. When considering the structure of a particular double-stranded DNA molecule, the sequence here is in the 5 'to 3' direction along the non-transcribed strand of DNA (ie, the strand having the same sequence as the mRNA), in the usual manner. You may list only. “Recombinant DNA molecule” means a DNA molecule that has undergone a molecular biological manipulation.

  By “isolated” nucleic acid molecule is meant a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. In certain instances, an “isolated” nucleic acid is a sequence that is naturally present at both ends of the nucleic acid encoding GAVE6 in the genomic DNA of the organism from which the nucleic acid is derived (ie, located at the 5 ′ and 3 ′ ends of the nucleic acid). Array) is not included. In various embodiments, the isolated GAVE6 nucleic acid molecule is a nucleic acid sequence of about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb or less, wherein the genetic DNA of the cell from which the nucleic acid was isolated. May include regions present at both ends of the nucleic acid molecule. Furthermore, an “isolated” nucleic acid molecule, such as a cDNA molecule, when produced by recombinant techniques, is substantially free of other intracellular materials or culture solutions and is chemically synthesized. Can be substantially free of chemical precursors or other chemicals.

  Nucleic acid molecules of the invention, eg, nucleic acid molecules having the nucleic acid sequence of SEQ ID NO: 1 or fragments thereof, or complementary to any of the nucleic acid sequences, or analogs or derivatives thereof are standard molecular biological It can be isolated using techniques and the sequence information disclosed herein. The GAVE6 nucleic acid molecule can be isolated by standard hybridization techniques and cloning techniques (eg, as described by Sambrook et al.) Using the full length or a portion of the nucleic acid sequence of SEQ ID NO: 1 as a hybridization probe. it can.

  The nucleic acid molecules of the present invention can be amplified according to standard PCR amplification techniques using cDNA, mRNA or genetic DNA as a template and appropriate oligonucleotide primers. Such a primer can be easily prepared using the information of SEQ ID NO: 1 and general experimental techniques. The nucleic acid so amplified is incorporated into a suitable vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GAVE6 nucleic acid sequences can be produced by standard synthetic methods, eg, automated DNA synthesizers.

An isolated nucleic acid molecule capable of hybridizing to GAVE6 DNA The present invention further relates to a hybridization probe capable of hybridizing under stringent hybridization conditions to a complementary hybridization probe capable of hybridizing to GAVE6 DNA, or a stringent high molecule. It includes isolated nucleic acid molecules that are capable of hybridizing to both under hybridization conditions. In particular, the present invention relates to a nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1 or a probe complementary to an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1 under stringent hybridization conditions. Includes isolated nucleic acid molecules capable of hybridizing.

A nucleic acid molecule is “hybridizable” to other nucleic acid molecules, eg, cDNA, genetic DNA or RNA, when a single-stranded nucleic acid molecule is suitable for other nucleic acid molecules at the appropriate temperature and solution ions. This is when it can be annealed under conditions of strength (see Sambrook et al., Supra). Temperature and ionic strength conditions determine the “stringency” of hybridization. For pre-screening of homologous nucleic acids, low stringency hybridization conditions with a T _m of 55 ° C., eg 5 × SSC, 0.1% SDS, 0.25%
Milk and no formamide; or 30% formamide, 5 × SSC, 0.5% SDS can be used. Intermediate stringency hybridization conditions correspond to higher T _m , eg, 40% formamide and 5 × or 6 × SSC. High stringency hybridization conditions correspond to the highest T _m , eg, 50% formamide, 5 × or 6 × SSC.

Hybridization requires two nucleic acids containing complementary sequences and depends on the stringency of hybridization, but there may be mismatches between bases. The appropriate stringency for hybridizing to a nucleic acid depends on the length of the nucleic acid and the degree of complementation, and can be varied as is well known in the art. The higher the degree of similarity or homology between two nucleic acid sequences, the higher the T _m for hybrids of nucleic acids containing those sequences. The relative stability of nucleic acid hybridization (corresponding to higher T _m ) decreases in the following RNA: RNA, DNA: RNA, DNA: DNA order. For hybrids longer than 100 nucleotides, a formula for calculating _Tm has been derived (see Sambrook et al., Supra, 9.50-0.51). For hybridization with shorter nucleic acids, ie oligonucleotides, the position of the mismatch becomes more important and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8). The minimum length of a hybridizable nucleic acid molecule is at least about 20 nucleotides; particularly at least about 30 nucleotides; more particularly at least about 40 nucleotides; even more particularly at least about 50 nucleotides; even more particularly at least about 60 nucleotides. It is. In particular, in an embodiment of the invention, the hybridizable nucleic acid molecule of the invention comprises at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000 Alternatively, it is 1100 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1, preferably the coding sequence, a sequence complementary thereto, or a fragment thereof.

As used herein, the phrase “hybridizes under stringent conditions” refers to nucleic acid sequences that are complementary to each other by at least 55%, 60%, 65%, 70%, and preferably 75% or more. It is intended to describe conditions for hybridization and washing, typically while remaining hybridized. Such stringent conditions are well known to those skilled in the art and are described in "Current Protocols in Molecular Biology", John Wiley.
& Sons, NY (1989), 6.3.1-6.3.6. Non-limiting examples of preferred stringent hybridization conditions include hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 50 × 65 ° C. with 0.2 × SSC, 0.1% SDS. Wash at least once. Preferably, an isolated nucleic acid molecule of the present invention is a naturally occurring nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 or its complementary sequence. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a natural nucleic acid sequence (eg, encoding a natural protein). One skilled in the art will understand that these conditions can be modified in view of sequence specific differences (eg, length, GC richness, etc.).

  The present invention is also intended to encompass nucleic acid fragments of GAVE6 which are diagnostic agents for GAVE6-like molecules with similar properties. This diagnostic fragment may be taken from any part containing the flanking sequence of the GAVE6 gene. This fragment can be used as a library probe by performing a known method.

  Furthermore, the nucleic acid molecules of the invention can comprise a portion of a nucleic acid sequence encoding GAVE6, which can be used, for example, as a probe or primer, or a fragment encoding a biologically active portion of GAVE6. Can be given. For example, such a fragment can include, but is not limited to, a region encoding from about 1 to about 14 amino acid residues of SEQ ID NO: 2. Nucleic acid sequences determined by cloning of the human GAVE6 gene create probes or primers for identification and / or cloning of GAVE6 homologues from other cell types, eg, other organs, or GAVE6 homologues from other mammals can do. This probe / primer typically comprises a substantially purified oligonucleotide. The oligonucleotide is typically at least about 12, preferably about 25, more preferably about 50, 75, 100, 125 of the sense or antisense of SEQ ID NO: 1 or a naturally occurring variant of SEQ ID NO: 1. , 150, 175, 200, 250, 300, 350, or 400 of nucleic acid sequences that hybridize under stringent conditions. Probes based on the human GAVE6 nucleic acid sequence can be used to detect transcripts or gene sequences encoding similar or identical proteins.

  As used herein, the term “fragment” or “portion” of an isolated nucleic acid molecule of the present invention means at least 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, It contains 175, 200, 250, 300, 350 or 400 contiguous nucleotides in length. To that end, a “fragment” of an isolated nucleic acid molecule of the present invention is not simply one or two nucleotides.

  Similarly, the term “fragment” or “portion” of a polypeptide of the invention comprises at least 9 consecutive amino acid residues. In particular, examples of “fragments” of the polypeptides of the invention include the epitope to which the GAVE6 antibody or fragment thereof binds.

A nucleic acid fragment encoding “a biologically active portion of GAVE6” is obtained by isolating a portion of SEQ ID NO: 1 encoding a polypeptide having a biological activity of GAVE6, and a polypeptide encoding a portion of GAVE6 protein Can be produced (eg, by in vitro recombinant expression methods) and evaluated by assessing the activity of a polypeptide encoding a portion of GAVE6. The present invention further includes a nucleic acid molecule that is different from the nucleic acid sequence of SEQ ID NO: 1 and encodes the same protein as the GAVE6 protein encoded by the nucleic acid sequence of SEQ ID NO: 1 due to the degeneracy of the genetic code. .

Homologous nucleic acid molecules The present invention further encompasses isolated nucleic acid molecules that are homologous to the GAVE6 DNA molecule, such as those that are homologous to the isolated nucleic acid molecule having the DNA sequence of SEQ ID NO: 1. Two DNA sequences are “substantially homologous” or “substantially similar” by at least about 50% (preferably at least about 75%, and more preferably at least about 90 or 95). %) Of nucleotides within the defined length of the DNA sequence. Substantially homologous sequences can be generated by standard software available in the sequence databank using default parameters or by Southern hybridization experiments, eg, under stringent conditions defined for a particular system. Can be identified. Defining appropriate hybridization conditions is within the ordinary skill in the art. See, for example, Maniatis et al., Supra; DNA Cloning, Volumes I and II, supra; Nucleic Acid Hybridization, supra. Furthermore, nucleic acid molecules encoding GAVE6 proteins (GAVE6 homologues) derived from other species and having a nucleic acid sequence different from human GAVE6 are also intended to be within the scope of the present invention.

Variants of nucleic acid molecules isolated according to the invention The invention further encompasses variants of the isolated nucleic acid molecules comprising the DNA sequence of SEQ ID NO: 1. Such variants are degenerate variants, alleles, or combinations thereof.

  Naturally-occurring allelic variants and homologues of the GAVE6 cDNA of the present invention are based on the identity of the human GAVE6 nucleic acid disclosed herein, and are stringent by using human cDNA or a portion thereof as a hybridization probe. Isolation by standard hybridization techniques under hybridization conditions.

  The term “corresponding to” means herein a sequence that is similar or homologous and the exact position is the same or different from the molecule for which similarity or homology was measured. As such, the term “corresponds to” means sequence similarity, not amino acid residue or nucleotide base numbering.

  Furthermore, due to the degenerate nature of the genetic code, the GAVE6 protein of the present invention can be encoded by many isolated nucleic acid molecules. “Degenerate” means the use of different three letter codons to determine the specific amino acid defined by the genetic code.

It is well known in the art that the following codons each encode a specific amino acid and can be used interchangeably:
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 of 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,
Termination codon UAA (Ocher) or UAG (Amber) or UGA (Opal).

  It should be understood that the codons specified above are of RNA sequence. The corresponding DNA codon uses T instead of U.

  In addition to the human GAVE6 nucleic acid sequence shown in SEQ ID NO: 1, those skilled in the art may have polymorphisms of DNA sequences that result in mutations in the amino acid sequence of GAVE6 in a biological population (eg, a human population) You will understand that. Such genetic polymorphisms in the GAVE6 gene may exist among individuals in a population due to natural allelic variation. An allele is one of a group of genes that occur alternatively at a given locus. As used herein, the terms “gene” and “recombinant gene” mean a nucleic acid molecule comprising an open reading frame encoding a GAVE6 protein, preferably a mammalian GAVE6 protein. As used herein, the phrase “allelic variant” refers to a nucleic acid sequence that occurs at the GAVE6 locus or a polypeptide encoded by that nucleic acid sequence. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be easily done with hybridization probes that identify the same locus in different individuals. Any and all nucleotide variants and resulting amino acid polymorphs or variants that are the result of natural allelic variation and that do not alter the functional activity of GAVE6 are intended to be within the scope of the present invention. Has been.

  Furthermore, variants of the isolated nucleic acid molecules of the present invention can be readily made by those skilled in the art using routine laboratory techniques, such as site-directed mutagenesis.

Antisense Nucleic Acid Sequences The present invention also relates to antisense nucleic acid molecules, ie molecules complementary to a sense nucleic acid encoding a protein, such as those complementary to the coding strand of a double stranded cDNA molecule, or to mRNA sequences. Complementary ones are included. Accordingly, antisense nucleic acids can form hydrogen bonds with sense nucleic acids. The antisense nucleic acid can be complementary to the full length or a portion of the GAVE6 coding strand, eg, the full length or partial protein coding region (or open reading frame). The antisense nucleic acid molecule can be antisense to the non-coding region of the coding strand of the nucleic acid sequence encoding GAVE6. Non-coding regions ("5 'and 3' untranslated regions") are 5 'and 3' sequences at both ends of the coding region and are not translated into amino acids.

  Given the coding strand sequence (eg, SEQ ID NO: 1) encoding GAVE6 disclosed herein, the antisense nucleic acid of the invention can be designed according to the rules of base pairing of Watson & Crick. The antisense nucleic acid molecule can be complementary to the full length of the coding region of GAVE6 mRNA, but more preferably is an oligonucleotide that is antisense to a coding region or non-coding region portion of GAVE6 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GAVE6 mRNA. Antisense oligonucleotides have, for example, a length of about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. The antisense nucleic acid of the present invention can be constructed using chemical synthesis and enzyme binding reactions by methods known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) are naturally occurring nucleotides or those formed to increase the biological stability of the molecule or between the antisense and sense nucleic acids. Chemical synthesis using a variety of chemically modified nucleotides designed to increase the physical stability of heavy chains (eg, phosphothioate derivatives, phosphoate derivatives and acridine substituted nucleotides can be used) can do.

Examples of modified nucleotides that can be used to make 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-galactosilk eosin, inosine, N ⁶ -iso Pentenyl adenine, 1-methyl guanine, 1-methyl inosine, 2,2-dimethyl guanine, 2-methyl adenine, 2-methyl guanine, 3-methyl cytosine, 5-methyl cytosine, N ⁶ -adenine, 7-methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl 2-thiouracil, beta-D-mannosyltransferase silk eosin, 5-methoxy-carboxymethyl uracil, 5-methoxy uracil, 2-methylthio--N ⁶ - isopentenyladenine, uracil-5-oxy acetic acid, Uibutokisoshin, pseudoephedrine uracil, Kueoshin, 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.

  As an alternative, antisense nucleic acids can be expressed in the form of an expression vector in which the nucleic acid is subcloned in the antisense direction (ie, the RNA transcribed from the inserted nucleic acid is in the antisense direction relative to the target nucleic acid of interest). And can be produced biologically. The antisense nucleic acid molecules of the present invention hybridize or bind to cellular mRNA and / or genetic DNA encoding GAVE6 to inhibit expression of the protein, eg, transcription and / or translation, Typically, it is administered to a patient or generated in situ. Normal nucleotide complementarity allows hybridization and forms a stable duplex or, for example, in the case of an antisense nucleic acid molecule that binds to a DNA duplex, the major groove of the double helix ) Binds to the DNA duplex or the control region of GAVE6 through specific interactions in

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

  The antisense nucleic acid molecule of the present invention can be an α-anomeric nucleic acid molecule. α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNAs whose strands run parallel to each other (Gaultier et al., Nucleic Acids Res (1987) 15: 6625-6641) . Antisense nucleic acid molecules can also be methylribonucleotides (Inoue et al., Nucleic Acids Res (1987) 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al., FEBS Lett (1987) 215 Volume: 327-330).

Ribozymes The present invention also encompasses ribozymes. Ribozymes are catalytic RNAs with ribonuclease activity that can cleave single-stranded nucleic acids that hybridize to the ribozyme, such as mRNA. To that end, ribozymes (eg, hammerhead ribozymes, Haselhoff et al., Nature (1988) 334: 585-591) can be used to catalytically cleave GAVE6 mRNA transcripts, resulting in: Inhibits translation of GAVE6 mRNA. A ribozyme having specificity for a nucleic acid encoding GAVE6 can be designed based on the nucleic acid sequence of GAVE6 DNA disclosed herein (for example, SEQ ID NO: 1). For example, Tetrahymena L-19 IVS RNA derivatives can be constructed such that the active site nucleic acid sequence is complementary to the nucleic acid sequence that is cleaved within the GAVE6 coding mRNA. See, for example, US 4,987,071 and 5,116,742. Alternatively, GAVE6 mRNA can be used to select a catalytic RNA having specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel et al., Science (1993) 261: 1411-1418.

Triple Helix Nucleic Acid Molecules and Peptide Nucleic Acids of the Invention The present invention also encompasses nucleic acid molecules that form triple helix structures. For example, GAVE6 gene expression targets a nucleic acid sequence complementary to the control region of GAVE6 (eg, GAVE6 promoter and / or enhancer) to form a triple helix structure that inhibits transcription of the target cell GAVE6 gene. Can be suppressed. In general, Helene, Anticancer Drug Des (1991) 6 (6): 569; Helene, Ann NY Acad Sci (1992) 660: 27; and Maher, Bioassays (1992) 14 Volume (Issue 12): see page 807.

  In certain embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety, or phosphate binding backbone, for example, to improve the stability, hybridization or solubility of the molecule. For example, the deoxyribose phosphate binding backbone of nucleic acids can be modified to create peptide nucleic acids (see Hyrup et al., Bioorganic & Medicinal Chemistry (1996) 4: 5). As used herein, a “peptide nucleic acid” or “PNAs” is a nucleic acid mimic, eg, a DNA mimic, in which the deoxyribose phosphate binding backbone of the nucleic acid is replaced with a pseudopeptide binding backbone. A kind of naturally occurring nucleobase means retained. The neutral backbone of PNA has been shown to allow specific hybridization with DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers is performed using standard solid phase peptide synthesis procedures (eg Hyrup et al. (1996) supra; Perry-O'Keefe et al., Proc Natl Acad Sci USA (1996) 93: 14670, As described in).

  GAVE6 PNA can be used for therapeutic and diagnostic purposes. For example, PNA can be used as an antisense or anti-gene agent for sequence-specific regulation of gene expression, eg, induces transcription, stops translation, or inhibits replication. GAVE6 PNA can also be used. For example, PNA can be used for analysis of single base pair mutations in genes, for example by artificial restriction enzymes (when used in combination with other enzymes, such as S1 nuclease, for example by PNA-directed PCR clamping). Hyrup et al. (1996) supra) or as a DNA sequence and probe or primer for hybridization (eg Hyrup et al. (1996) supra; Perry-O'Keefe et al. Supra).

  In other embodiments, the PAVE of GAVE6 is, for example, by linking a lipophilic or other helper group to the PNA to promote stability, specificity or cellular uptake, or PNA Modifications can be made by forming a DNA chimera or by using liposomes or other techniques known in the art for drug delivery. Synthesis of PNA-DNA chimeras is described in Hyrup et al. (1996) above; 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 Med Chem Lett (1975) 5: 1119.

In addition, the present invention includes an isolated polypeptide comprising the amino acid sequence of FIG. 2 (SEQ ID NO: 2), a variant thereof, a fragment thereof or an analogue or derivative thereof.

  An isolated nucleic acid molecule encoding a GAVE6 protein having a sequence different from SEQ ID NO: 2, such as a variant, introduces one or more nucleotide substitutions, additions or deletions into the nucleic acid sequence of SEQ ID NO: 1 By way of example, one or more amino acid substitutions, additions or deletions can be made in the encoded protein.

  In certain embodiments, for a variant of the GAVE6 protein, (1) the ability to form a protein: protein interaction with a protein in the GAVE6 signaling pathway; (2) the ability to bind a GAVE6 ligand; or (3) a cell The ability to bind to an internal target protein can be measured. In still other embodiments, the ability of GAVE6 variants to modulate cell proliferation or cell differentiation can be measured.

  Native GAVE6 protein can be isolated and purified from cell or tissue material using standard protein purification techniques by an appropriate purification procedure. As an alternative, the GAVE6 protein can be readily produced by recombinant DNA technology. In addition, another alternative encompassed by the present invention is chemical synthesis using standard peptide synthesis techniques for GAVE6 proteins or polypeptides.

An “isolated” or “purified” protein, or biologically active portion thereof, refers to any intracellular material or other contaminating protein derived from the cell or tissue material from which the GAVE6 protein is isolated. Or in the case of chemical synthesis is substantially free of chemical precursors or other chemicals. The phrase “substantially free of intracellular material” includes preparations of the GAVE6 protein from which the GAVE6 protein can be isolated or separated from cellular components of cells produced by recombinant methods. Thus, GAVE6 protein substantially free of intracellular material includes non-GAVE6 protein (also referred to herein as “contaminating protein”) at about 30%, 20%, 10% or 5% or less ( Includes (by dry weight). When the GAVE6 protein or biologically active portion thereof is produced by recombinant methods, it is also preferably substantially free of culture broth, ie, the culture broth is about 20% of the volume of the protein preparation, Contains 10% or 5% or less. When the GAVE6 protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., chemical precursors or other chemicals involved in the synthesis of the protein Has been separated from. Accordingly, such GAVE6 protein preparations contain only about 30%, 20%, 10% or 5% or less (by dry weight) of chemical precursors or non-GAVE6 chemicals.

  The biologically active portion or fragment of the GAVE6 protein includes an amino acid sequence that is sufficiently identical to or derived from the amino acid sequence of the GAVE6 protein (eg, the amino acid sequence of SEQ ID NO: 2), and is a full-length GAVE6 protein Peptides comprising an amino acid sequence comprising a shorter amino acid sequence and exhibiting at least one activity of the GAVE6 protein are included. Typically, the biologically active portion comprises at least one domain or motif having the activity of GAVE6 protein. The portion of the biologically active GAVE6 protein is a polypeptide, i.e., for example, 10, 25, 50, 100 or more amino acids in length. In particular, the biologically active polypeptide comprises one or more identified GAVE6 structural domains.

  In addition, other biologically active portions, in which other regions of the protein are deleted, can be produced by recombinant techniques, and can be one or more of the native GAVE6 proteins Are evaluated for functional activity.

  Other useful GAVE6 proteins are substantially the same as SEQ ID NO: 2, maintain the functional activity of the protein of SEQ ID NO: 2 and still differ in amino acid sequence due to natural allelic variation or mutation Is. For example, such GAVE6 proteins and polypeptides have at least one biological activity described herein.

  Thus, a useful GAVE6 protein comprises an amino acid sequence that is at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2, The protein maintains the functional activity of the GAVE6 protein of SEQ ID NO: 2. In a special embodiment, the GAVE6 protein retains the functional activity of the GAVE6 protein of SEQ ID NO: 2.

  To determine the percent homology of two amino acid sequences or two nucleic acids, the sequences are aligned so that an optimal comparison can be made (eg, the first nucleic acid sequence or amino acid sequence includes a second nucleic acid sequence). Alternatively, gaps can be inserted to obtain an optimal sequence of amino acid sequences). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When the position of the first sequence is the same amino acid residue or nucleotide as the corresponding position of the second sequence, these molecules are judged to be identical at that position. The percent homology between two sequences is a function of the number of identical positions common in those sequences (ie, homology (percent) = number of identical positions / total number of positions (eg, overlap) Position) × 100). In one embodiment, the two sequences are the same length.

  The determination of percent homology between two sequences can be accomplished by a mathematical algorithm. In particular, without limitation, examples of mathematical algorithms that can be used to compare two sequences include: Karlin et al., Proc Natl Acad Sci USA (1990) 87: 2264, and a modified method by Karlin et al., Proc Natl Acad Sci USA (1993) 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J Mol Bio (1990) 215: 403. The BLAST nucleotide search can be performed using the NBLAST program. For example, in order to obtain a nucleic acid sequence homologous to the GAVE6 nucleic acid molecule of the present invention, a score = 100 and a word length = 12. The BLAST protein search can be performed using the XBLAST program. In order to obtain an amino acid sequence homologous to the GAVE6 protein molecule of the present invention, the score = 50 and the word length = 3. To obtain a gapped sequence for comparison, a gapped BLAST (Gapped BLAST) can be used as described in Altschul et al., Nucleic Acids Res (1997) 25: 3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. Altschul et al. (1997), above. When using 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.). .

  Other non-limiting examples of mathematical algorithms that can be used for sequence comparison include the Myers et al. Algorithm, CABIOS (1988) 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, a 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 methods similar to those described above, with or without allowing gaps. When calculating percent identity, only exact matches are counted.

  The invention further encompasses GAVE6 chimeric or fusion proteins. As used herein, a GAVE6 “chimeric protein” or “fusion protein” includes a GAVE6 polypeptide operably linked to a non-GAVE6 polypeptide. “GAVE6 polypeptide” means a polypeptide having an amino acid sequence corresponding to GAVE6. A “non-GAVE6 polypeptide” is a polypeptide having an amino acid sequence corresponding to a protein that is not substantially identical to GAVE6 (eg, a protein that is different from GAVE6 protein and is derived from the same or a different organism). means. In a GAVE6 fusion protein, the GAVE6 polypeptide can correspond to all or a portion of the GAVE6 protein, and is preferably a portion of at least one biologically active GAVE6 protein. In the fusion protein, the term “operably linked” means that the GAVE6 and non-GAVE6 polypeptides are linked in-frame to each other. The non-GAVE6 polypeptide can be linked to the N-terminus or C-terminus of the GAVE6 polypeptide. One useful fusion protein is GST-GAVE6, where the GAVE6 sequence is linked to the C-terminus of glutathione-S-transferase (GST). Such fusion proteins can facilitate the purification of recombinant GAVE6.

In other embodiments, the fusion proteins of the invention include GAVE6-immunoglobulin fusion proteins, in which all or part of GAVE6 is linked to a sequence derived from a member of the immunoglobulin protein family. The GAVE6-immunoglobulin fusion protein of the present invention can be incorporated into a pharmaceutical composition to inhibit the interaction between GAVE6 ligand and GAVE6 protein on the cell surface and to suppress GAVE6-mediated signaling in vivo. Can be administered to patients. The GAVE6-immunoglobulin fusion protein can be used to influence the bioavailability of a cognate ligand to GAVE6. Inhibition of the GAVE6 ligand-GAVE6 interaction is useful therapeutically and can be used to modulate (eg, promote or inhibit) proliferative and differentiation disorders and cell viability. Furthermore, the GAVE6-immunoglobulin fusion protein of the present invention is used as an immunogen to allow patients to make anti-GAVE6 antibodies, for purification of GAVE6 ligand, and for the identification of molecules that inhibit the interaction of GAVE6 ligand and GAVE6. Can be used.

  In certain embodiments, the GAVE6 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 frame by universal techniques: for example, blunt-ended or stagger-ended for ligation. ), Restriction enzyme cleavage to provide appropriate ends, filling-in cohesive ends if necessary, alkaline phosphatase treatment to avoid undesired binding, and enzymatic A ligation reaction is mentioned. In other embodiments, the fusion gene can be synthesized by general techniques including automated DNA synthesizers. Instead, PCR amplification of gene fragments is performed using an anchor primer complementary to the overhang between two consecutive gene fragments, annealing and re-amplifying to generate a chimeric gene sequence. (See, eg, Ausubel et al., Supra). In many more expression vectors, commercially available ones that already encode a fusion moiety (eg, a GST polypeptide) can be used. A nucleic acid encoding GAVE6 can be cloned in such an expression vector such that the fusion moiety is in frame with the GAVE6 protein.

Variants As explained above, the present invention is further introduced into the amino acid sequence of SEQ ID NO: 2 by standard techniques such as variants of the GAVE6 protein, eg, site-directed mutagenesis and PCR-mediated mutagenesis. Mutants. Furthermore, conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, one or more amino acids can be replaced by other amino acids having the same polarity, which acts as a functional equivalent, resulting in a silent substitution. An amino acid substitution in the amino acid sequence of the polypeptide of the present invention is selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic rings include 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 substitution will not affect the apparent molecular weight or isoelectric point that can be measured by polyacrylamide gel electrophoresis.

Particularly preferred is the substitution of Lys instead of Arg and its reverse substitution to maintain the positive charge; the substitution of Glu instead of Asp and its reverse substitution to maintain the negative charge; Ser instead of Thr Substitution to maintains the free OH group; substitution to Gln instead of Asn maintains the free NH ₂ group.

  Furthermore, amino acid substitutions may be introduced to replace amino acids having particularly favorable properties. For example, Cys can be introduced for sites that may form disulfide bridges with other Cys. His can in particular introduce a “catalytic” site (ie His acts as an acid or base and is the most common amino acid in biochemical catalysis). Due to its special planar structure, Pro can be introduced to form a β-turn in the protein structure.

Mutations can also be introduced randomly along the entire length or portion of the sequence encoding GAVE6 (eg, saturation mutagenesis), and the resulting mutants are suddenly retained activity. It can be used when screening the biological activity of GAVE6 to identify mutants. After mutation, the protein encoded thereby can be expressed recombinantly and the activity of the protein is measured.

  The variants of the invention can function as GAVE6 agonists (mimetics) or GAVE6 antagonists. GAVE6 protein variants can be generated by mutation methods, such as individual point mutations or shortening of the GAVE6 protein. An agonist of the GAVE6 protein can retain substantially the same or some combination thereof with the biological activity of a naturally derived GAVE6 protein. For example, antagonists of the GAVE6 protein competitively bind downstream or upstream of members of the cell signaling cascade that comprise the GAVE6 protein, thereby inhibiting one or more activities of the naturally-occurring GAVE6 protein. In this way, special biological activity can be elicited by treatment with variants with limited function. Treatment of patients with variants having some combination of naturally occurring GAVE6 protein activity can have fewer side effects compared to treatment with naturally occurring GAVE6 protein.

  GAVE6 protein variants that function as GAVE6 agonists (mimetics) or GAVE6 antagonists can be identified in association with GAVE6 agonist or antagonist activity from screening combinatorial libraries of GAVE6 protein variants (eg, truncation variants). In one embodiment, a variegated library of GAVE6 variants can be generated by combinatorial mutagenesis at the nucleic acid level, which is encoded by a variegated gene library. A variegated library of GAVE6 variants can be used, for example, to enzymatically mix synthetic oligonucleotides into gene sequences such that a degenerate set of potential GAVE6 sequences is expressed as individual polypeptides. Binding, or alternatively, can be made as a collection of larger fusion proteins (eg, for phage display) containing a collection of GAVE6 sequences therein. There are a variety of methods that can be used to generate a library of potential GAVE6 variants from a degenerate oligonucleotide sequence. Chemical synthesis of the degenerate gene sequence is performed with an automated DNA synthesizer, and the synthesized gene is then incorporated into an appropriate expression vector. The use of a degenerate set of genes allows for the supply of all of the sequences that encode the desired collection of potential GAVE6 sequences in one hybrid. Methods for synthesizing degenerate oligonucleotides are known in the art (eg, 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 Acid Res (1983) 11: 477).

  In addition, to produce a gene library that produces a collection of variegated GAVE6 fragments for screening and subsequent selection of mutants of the GAVE6 protein, a library of fragments of sequences encoding the GAVE6 protein is generated. Can be used. In one embodiment, a library of sequence fragments is obtained by subjecting a double-stranded PCR fragment encoding GAVE6 to nuclease treatment under conditions that produce only about one nicking per molecule, Denaturing the DNA, re-naturing the DNA to form a double-stranded DNA that can contain sense / antisense from products with different nicks, and regenerate by S1 nuclease treatment The single-stranded portion can be removed from the resulting duplex and the resulting fragment library can be incorporated into an expression vector. By such methods, expression libraries can be derived which encode various sizes of internal fragments of the GAVE6 protein and the N-terminus.

Various methods for screening gene products of combinatorial libraries created by point mutation methods or shortening and for screening cDNA libraries for gene products having selected properties Are known in the art. Such techniques can be applied to rapid screening of gene libraries generated by combinatorial mutagenesis of GAVE6 protein. The most widely used techniques can be applied to high-throughput analysis of large gene libraries, typically obtained by cloning the gene library into an expression vector capable of replicating and then obtaining the appropriate cells Techniques that include transforming with a library of vectors and expressing the combinatorial gene under conditions that can detect the desired activity that facilitates isolation of the vector encoding the gene whose product can be detected are included. Recursive ensemble mutagenesis (REM), a technique that increases the frequency of functional mutants in a library, should be used in combination with screening assays to identify GAVE6 variants (Arkin et al., Proc Natl Acad Sci USA (1992) 89: 7811-7815; Delgrave et al., Protein Engineering (1993) 6 (3): 327-331).

Analogs and Derivatives of GAVE6 Furthermore, the present invention also includes analogs and derivatives of GAVE6 produced by chemical modification methods. The GAVE6 protein of the invention can be derivatized by linking one or more chemical moieties to the protein moiety.

Chemical moieties suitable for derivatization Chemical moieties suitable for derivatization can be selected from water soluble polymers that prevent the GAVE6 analog or derivative from precipitating in an aqueous environment, such as a physiological environment. In some cases, the polymer will be pharmaceutically acceptable. One skilled in the art will consider whether the polymer / component conjugate is used therapeutically (if so, by considering the desired dosage, circulation time, resistance to proteolysis, and other considerations). The desired polymer could be selected. GAVE6 can be confirmed using the assay method provided here. Examples of water-soluble polymers used herein include, but are not limited to, polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1, 3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer), dextran, poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer , Polyoxyethylenated polyol or polyvinyl alcohol. Polyethylene glycol propionaldehyde is advantageous in production because of its stability in water.

  The polymer can be of any molecular weight and can be unbranched or branched. For polyethylene glycol, the preferred molecular weight is between about 2 kDa and about 100 kDa for ease of manufacture and operation (the term “about” refers to some molecules in the preparation of polyethylene glycol, Greater than the molecular weight and some are smaller). Other sizes may also affect the desired therapeutic profile (eg, desired duration of sustained release, effects on biological activity, if any, ease of manipulation, degree or lack of antigenicity, and other , Known effects of polyethylene glycol on therapeutic proteins or analogs).

The number of polymer molecules that adhere to GAVE6 may vary, and one skilled in the art will be able to confirm the effect on function. It can be a single substitution or a combination of two, three, four substitutions or substitutions with the same or different chemical moieties (eg, polymers, polyethylene glycols of different molecular weights). The ratio of polymers to GAVE6 molecules will vary depending on their concentration in the reaction. In general, the optimal ratio (meaning reaction efficiency in which there is no excess of single or multiple unreacted components and polymer present) is the desired degree of derivatization (eg, mono, di, tri). Etc.), the molecular weight of the chosen polymer, whether the polymer is branched or unbranched, and the reaction conditions.

  A polyethylene glycol molecule (or other chemical moiety) should be attached to GAVE6 in view of its effect on the functional or antigenic domains of GAVE6. There are many conjugation methods available to those skilled in the art, for example EP0401384 (coupling of PEG to G-CSF), incorporated herein by reference, or 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 to an amino acid via a reactive group such as a free amino or carboxyl group. The reactive group is a group to which an activated polyethylene glycol molecule can bind. Amino acids having a free amino group include lysine residues and N-terminal amino acid residues; amino acids having a free carboxyl group include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment to an amino group, such as attachment to the N-terminus or lysine group.

In particular, GAVE6 in which the N-terminus is chemically modified is desirable. To illustrate this composition, polyethylene glycol is used to vary the various polyethylene glycol molecules (molecular weight, branching state, etc.), the ratio of polyethylene glycol molecules to GAVE6 molecules in the reaction mixture, the pegylation reaction to be performed. And the method for obtaining a protein PEGylated at the selected N-terminus. The method of obtaining a preparation PEGylated at the N-terminus (ie separating this part from other single pegylation moieties, if necessary) involves the removal of a substance PEGylated at the N-terminus from a collection of pegylated protein molecules. This is due to purification. Selective N-terminal chemical modification can be achieved by reductive alkylation that results in different reactivity of different types of primary amino groups (lysine to the N-terminus) available for derivatization of GAVE6. Under appropriate reaction conditions, substantially selective derivatization with a polymer containing a carbonyl group at the N-terminus of GAVE6 is achieved. For example, by selectively carrying out the reaction at a pH that makes it possible to advantageously use _a pKa that differs between the ε-amino group of the lysine residue and the N-terminal α-amino group of GAVE6, the N-terminus of GAVE6 Can be pegylated. Such selective derivatization can control the binding of a water-soluble polymer to GAVE6; conjugation with the polymer occurs primarily at the N-terminus of GAVE6 and modifies other reactive groups (eg, lysine side chains). Of the amino group) does not substantially occur. Using reductive alkylation, the water-soluble polymer can be of the type described above and should have one reactive aldehyde for conjugation with GAVE6. Polyethylene glycol propionaldehyde containing one reactive aldehyde can be used.

An antibody of GAVE6, a variant thereof, a fragment thereof, or an analogue or derivative thereof The isolated GAVE6 protein or portion or fragment thereof binds to GAVE6 using standard techniques for producing polyclonal and monoclonal antibodies Can be used as an immunogen to generate antibodies. As used herein, the term “antibody” refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, ie, an antigen binding site that specifically binds an antigen such as GAVE6 or a fragment thereof. Means a molecule. A molecule that specifically binds to GAVE6 is a molecule that binds to GAVE6 but does not substantially bind to other molecules in a specimen (eg, a biological sample that naturally contains GAVE6). Examples of immunologically active portions of immunoglobulin molecules include F _(ab) and F _{(ab ′) 2} fragments, which can be generated by digesting antibodies with enzymes such as pepsin.

  The present invention provides polyclonal, monoclonal, and chimeric antibodies that use GAVE6, a variant thereof, a fragment or similar substance thereof, or a derivative thereof as an immunogen. For the purpose of the treatment of human diseases or disorders, preferably chimeric antibodies are used, in which human or humanized antibodies have an immune response against themselves, in particular an allergic response, compared to xenogeneic antibodies. It is because it is hard to cause.

  The full length GAVE6 protein can be used, and alternatively, the present invention also provides an antigenic peptide fragment of GAVE6 that can be used as an immunogen. The antigenic peptide of GAVE6 contains at least 8 (preferably 10, 15, 20, 30 or more) amino acid residues of the amino acid sequence of SEQ ID NO: 2, and the antibody produced against the peptide is GAVE6 Also included are epitopes of GAVE6 that form specific immune complexes with.

  A GAVE6 immunogen is typically used to immunize a suitable animal species (eg, rabbit, goat, mouse or other mammal) with the immunogen to produce antibodies. Suitable immunogenic preparations include, for example, recombinantly expressed GAVE6 protein or chemically synthesized GAVE6 polypeptide. The preparation can further include Freund's complete or incomplete adjuvant or similar immunostimulatory agent. Immunization of appropriate animals with an immunogenic GAVE6 preparation induces a polyclonal anti-GAVE6 antibody response.

  The antibody of the present invention may be a monoclonal antibody, a polyclonal antibody or a chimeric antibody. As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to a collection of antibody molecules that contain only one type of antigen binding site capable of immunoreacting with a particular epitope of GAVE6. Thus, monoclonal antibody compositions typically exhibit a single binding affinity for a particular GAVE6 protein epitope.

  Polyclonal anti-GAVE6 antibodies can be produced by immunizing a suitable animal with a GAVE6 immunogen as described above. The titer of anti-GAVE6 antibody in the immunized animal can be monitored throughout the experiment by standard techniques, eg, enzyme linked immunosorbent assay (ELISA) using immobilized GAVE6. If necessary, antibody molecules against GAVE6 can be isolated from mammals (eg, blood) and further purified by well-known methods for obtaining IgG fractions, such as protein A chromatography. it can. Antibody producing cells can be harvested from immunized animals at an appropriate time after immunization, eg, when the titer of anti-GAVE6 antibody is at the highest value, and can be obtained using standard techniques (eg, hybridomas first described by Kohler et al. Technology, Nature (1975) 256: 495-497, human B cell hybridoma technology (Kohler et al., Immunol Today (1983) 4: 72), EBV hybridoma technology (Cole et al., Monoclonal Antibody and Cancer Therapy ( 1985) Alan R. Liss, Inc., 77-96), or trioma technology) can be used to produce monoclonal antibodies. Techniques for producing hybridomas are well known (generally referred to are Current Protocols in Immunology (1994) Coligan et al., John Wiley & Sons, Inc., New York NY). Briefly, an immortal cell line (typically myeloma) is fused with a mammalian white blood cell (typically a spleen cell) immunized with the above-mentioned GAVE6 immunogen, and the resulting hybridoma is cultured. Screening is performed to identify hybridoma cells producing monoclonal antibodies that bind to GAVE6.

  Any of the well-known protocols for fusing leukocytes and immortalized cell lines can be employed for the purpose of generating anti-GAVE6 monoclonal antibodies. (Eg, Current Protocols in Immunology, supra; Galfre et al., Nature (1977) 266: 550-552; Kenneth, in Monoclonal Antibodies, A New Dimension In Biological Analyzes, Plenum Publishing Corp., New York NY (1980 And Lerner, Yale J Biol Med (1981) 54: 387-402). Furthermore, those skilled in the art will appreciate that there are many variations of such methods available. Typically, immortalized cell lines (eg, myeloma cell lines) are derived from the same mammal from which the leukocytes were obtained. For example, murine hybridomas are sensitive to leukocytes from mice immunized with the immunogenic preparations of the present invention and an immortalized mouse cell line (eg, culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”)). And a myeloma cell line). Any of a number of myeloma cell lines can be used as fusion partners according to standard techniques, examples include P3-NSl / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Agl4 myeloma. Cell lines. Such myeloma cell lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused with mouse spleen cells using polyethylene glycol (“PEG”). The hybridoma cells obtained as a result of the fusion are then selected using HAT medium that kills the non-fused and non-productively fused myeloma cells (the unfused spleen cells are not transformed and after a few days. To die). The hybridoma cell producing the monoclonal antibody of the present invention is screened for the presence or absence of an antibody that binds the hybridoma culture supernatant to GAVE6, for example, using a standard ELISA assay.

  As an alternative to preparing hybridomas that secrete monoclonal antibodies, monoclonal anti-GAVE6 antibodies are obtained by combining a recombinant combinatorial immunoglobulin library (eg, an antibody phage display library) with a single immunoglobulin library member that binds to GAVE6. Identification and isolation can be done by screening with GAVE6 so that it can be released. Commercially available kits for creating and screening phage display libraries are available (eg, Pharmacia recombinant phage antibody system, Catalog No. 27-9400-01; and Stratagene “SURFZAP” phage display kit, Catalog No. 240612).

  In addition, examples of methods and reagents that can be used in particular when creating and screening antibody display libraries are found, for example, in the following literature: US 5,223,409; WO 92/18619; WO91 / 17271; WO92 / 20791; WO92 / 01679; WO92 / 01047; WO92 / 09690; WO90 / 02809; Fuchs et al., Bio / Technology (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-GAVE6 antibodies, such as chimeric antibodies, and humanized monoclonal antibodies comprising human and non-human portions can be generated using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be made by recombinant DNA techniques known in the art. Examples of such techniques are those described in the following references: WO87 / 02671; EP184,187; EP171,496; EP173,494; WO86 / 01533; US4,816,567; EP125,023; Better et al., Science. (1988) 240: 1041-1043; Liu et al., Proc Natl Acad Sci USA (1987) 84: 3439-3443; Lin et al., J Immunol (1987) 139: 3521-3526; Sun Proc Natl Acad Sci USA (1987) 84: 214-218; Nishimura et al., Canc 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 Patent 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. Volume: 4053-4060.

Fully humanized antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced by transgenic mice that cannot express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes. Transgenic mice are immunized in the usual manner with a selected antigen, eg, all or part of GAVE6. Monoclonal antibodies against the antigen can be obtained by general-purpose hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice undergo rearrangement during B cell differentiation, followed by class switching and somatic mutation. Such epitopes are then used to identify antibodies that inhibit GAVE6 activity. The heavy and light chains of the non-humanized antibody are cloned and used to create phage display Fab fragments. For example, the heavy chain gene can be cloned into a plasmid vector such that the heavy chain can be secreted from the microorganism. The light chain gene can be cloned into a phage coat protein gene so that the light chain can be expressed on the surface of the phage. A repertoire (random collection) of human light chains fused to phage can be used to infect microorganisms that express non-human heavy chains. The resulting progeny phage displays a hybrid antibody (human light chain / non-human heavy chain). The selected antigen is used for panning screening to select phage that bind to the selected antigen. Several iterations of selection will be required to identify such phage.

  Human light chain genes can be isolated from selected phage that bind to the selected antigen. The selected human light chain gene is then used as follows to guide selection of the human heavy chain gene. The selected human light chain gene is incorporated into a vector for microbial expression. Microorganisms expressing the selected human light chain are infected with a repertoire of human heavy chains fused to the phage. The resulting progeny phage displays human antibodies (human light chain / human heavy chain).

The selected antigen is then used for panning screening to select for phage that bind to the selected antigen. The selected phage displays a fully human antibody that recognizes the same epitope recognized by the initially selected non-human monoclonal antibody. Genes encoding both heavy and light chains have been isolated and can be further manipulated to produce human antibodies. This technique is described by Jespers et al. (Bio / Technology
(1994) 12: 899-903).

  Anti-GAVE6 antibodies (eg, monoclonal antibodies) can be used for isolation of GAVE6 by standard techniques (eg, affinity chromatography or immunoprecipitation). The anti-GAVE6 antibody can efficiently purify GAVE6 produced from a cell derived from naturally-derived GAVE6 and produced by a recombinant method expressed in a host cell. Furthermore, the anti-GAVE6 antibody can be used to detect GAVE6 protein (for example, from a cell lysate or cell supernatant) for the purpose of examining the expression pattern and amount of GAVE6 protein. Anti-GAVE6 antibodies can be used diagnostically to monitor the amount of protein in a tissue as part of a clinical laboratory procedure (eg, to determine the effectiveness of a given treatment method). Detection can be facilitated by a conjugate of an antibody and a detectable substance as shown below.

Detectable labels In some cases, isolated nucleic acid molecules of the invention, polypeptides of the invention and antibodies of the invention, and fragments thereof can be detectably labeled. Suitable labels include enzymes, fluorescent dyes (for example, several fluorescent dyes such as fluorescent isothiocyanate (FITC), phycoerythrin (PE), Texas Red (TR), rhodamine, free or chelating lanthanides. Salts, in particular Eu ³⁺ ), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (eg biotin), bioluminescent materials, and chemiluminescent agents. When control markers are used, the same or different labels can be used for the receptor and control markers.

Radiolabels (eg, the following isotopes: ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ^l31 I, and ^l86 Re) In the example where is used, known currently available measurement methods can be used. Examples using enzyme labels can be detected by colorimetric, spectroscopic, fluorescent spectroscopic, amperometric, or gas quantification methods known in the art and currently available.

  Direct labeling is one example of a label that can be used according to the present invention. A direct label is defined as a whole and can be detected in its native state with the aid of the naked eye or an optical filter and / or applied stimuli such as UV light that enhances fluorescence. Examples of colored labels that can be used according to the present invention include metallic sol particles such as gold sol particles described by Leuvering (US 4,313,734); described by Grinbau et al (US 4,373,932) and May et al (WO 88/08534). Dye sol particles as described above; dyes latex described by May, supra, Snyder (EP-A0280559 and 0281327); or dyes encapsulated in liposomes described in Campbell et al. (US 4,703,017). Other direct labels include radioactive nucleotides, fluorescent moieties, or chromogenic moieties. In addition to such direct labeling tools, indirect labels include enzymes and can be used in accordance with the present invention. Various types of enzyme-linked immunoassays are well known in the art, such as alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, and others Eva Engvall, discussed in detail in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70, 419-439, 1980 and US 4,857,453.

  Other labeling methods used in the present invention include magnetic beads or magnetic resonance imaging labels.

In other embodiments, a phosphorylation site for ³² P labeling can be created in an isolated polypeptide of the invention, an antibody of the invention, or a fragment thereof, such as EP0372707 (application number by Sidney Pestka). 89311108.8) or US5,459,240 (issued October 17, 1995) granted to Foxwell et al.

As exemplified herein, proteins including antibodies can be labeled with metabolic labels. Metabolic labeling is performed in vitro by culturing cells expressing the protein in the presence of a culture medium supplemented with a metabolic label such as [ ³⁵ S] -methionine or [ ³² P] -orthophosphate. In addition to metabolic (ie, biosynthetic) labeling with [ ³⁵ S] -methionine, the present invention further includes [ ¹⁴ C] -amino acids and [ ³ H] -amino acids (substituted with tritium at non-fragile positions). Labeling with is also included.

Recombinant expression vectors and host cells Another aspect of the present invention relates to vectors, preferably expression vectors, comprising nucleic acids encoding GAVE6 (or portions thereof). As explained above, one type of vector is a “plasmid”, meaning a circular double-stranded DNA loop to which additional DNA fragments are linked. Another type of vector is a viral vector, in which additional DNA fragments are linked to the viral genome. Certain vectors are capable of autonomous replication in a host cell (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell when introduced into the host cell and replicated along with the host genome. Furthermore, the expression vector can express a gene operably linked thereto. In general, expression vectors utilized in recombinant DNA technology are often in the form of plasmids (vectors). However, the present invention is intended to include other expression vectors that provide equal function, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses).

  The recombinant expression vector of the present invention includes a nucleic acid molecule of the present invention suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vector of the present invention includes one or more control sequences, selected based on the host cell used for expression and operably linked to the nucleic acid to be expressed. In a recombinant expression vector, “operably linked” means that the nucleic acid sequence of interest is linked to a regulatory sequence in such a way that the nucleic acid sequence can be expressed. (Eg, in an in vitro transcription / translation system or in a host cell where the vector has been introduced into the host cell). The term “regulatory sequence” refers to 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). Control sequences include those that direct constitutive expression of the nucleic acid sequence in many types of host cells (eg, organ-specific control sequences). One of skill in the art will understand that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression 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 acid sequences described herein (eg, GAVE6 protein, GAVE6 variants, fusion proteins, etc.).

  The recombinant expression vector of the present invention expresses GAVE6 in prokaryotic or eukaryotic cells (eg, bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells). Can be designed to do. Suitable host cells are discussed by Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, eg, using phage regulatory elements and proteins (eg, T7 promoter and / or T7 polymerase).

  Expression of proteins in prokaryotes can often be performed in E. coli with constitutive or inducible promoters that express fused or non-fused proteins. A fusion vector adds several amino acids to the protein encoded therein, which are usually added to the amino terminus of the recombinant protein. Such fusion vectors typically serve as a ligand for three purposes: 1) facilitating expression of the recombinant protein, 2) increasing solubility of the recombinant protein; and 3) affinity purification. Used to facilitate protein purification. Often, in fusion expression vectors, a proteolytic site is introduced at the junction of the fusion moiety and the recombinant protein, allowing separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes have cognate recognition sequences, including active 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). And glutathione 5-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant protein of interest, 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: Methods in Enzymology, Academic Press, San Diego, California (1990) 185: 60-89). Expression of the gene of interest from the pTrc vector relies on transcription from a hybrid trp-lac fusion promoter by host RNA polymerase.

  One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host that lacks the ability to proteolyze the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology, Academic Press San Diego, California (1990) 185: 119-128). Another strategy is to change the nucleic acid sequence of the nucleic acid molecule that is incorporated into the expression vector into codons that are advantageously used in E. coli (Wada et al., Nucleic Acids Res (1992 20): 2111-2118). Such conversion of the nucleic acid sequence of the present invention can be performed by standard DNA synthesis techniques.

  In another embodiment, the GAVE6 expression vector is a yeast expression vector. Examples of expression vectors in yeast such as Saccharomyces cerevisiae include pYepSecl (Baldari et al., EMBO J (1987) 6: 229-234), pMFa (Kurjan et al., 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) included.

  As an alternative, GAVE6 can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors can be used for protein expression in cultured insect cells (eg, Sf 9 cells) and include the pAc series (Smith et al., 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 can be expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors used herein include, but are in no way limited to, pCDM8 (Seed, Nature (1987) 329: 840) and pMT2PC (Kaufman et al., EMBO J (1987) 6 : Pp. 187-195). When using mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus type 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for prokaryotic and eukaryotic cells, see Sambrook et al., Chapters 16 and 17 above.

  In other embodiments, the recombinant mammalian expression vectors of the invention can be directed to express more of the nucleic acid in a particular cell type (eg, because an organ-specific regulatory element expresses the nucleic acid). Can be used). Organ-specific regulatory elements are known in the art. Non-limiting examples of suitable organ specific promoters include albumin promoter (liver specific; Pinkert et al., Genes Dev (1987) 1: 268-277), lymphocyte specific promoter (Calame et al., Adv Immunol ( 1988) 43: 235-275), in particular T cell receptors (Winoto et al., EMBO J (1989) 8: 729-733) and immunoglobulins (Banerji et al., Cell (1983) 33: 729-740; Queen et al., Cell (1983) 33: 741-748), neuron-specific promoters (eg neurofilament promoter; Byrne et al., Proc Natl Acad Sci USA (1989) 86: 5473-5477), pancreas-specific promoters (Edlund et al., Science (1985) 230: 912-916) and mammary gland specific promoters (eg milk whey promoter; US 4,873,316 and EP 264,166) It is. Growth control promoters are also included; for example, the rodent hox promoter (Kessel et al., Science (1990) 249: 374-379) and the alpha fetoprotein promoter (Campes et al., Genes Dev (1989) 3): 537-546).

The present invention further provides a recombinant expression vector comprising the DNA molecule of the present invention cloned into the expression vector in the direction of antisense. That is, the DNA molecule expresses an RNA molecule that is antisense to GAVE6 mRNA (by transcription of the DNA molecule).
Is operably linked to the control sequence in such a way that Control sequences operably linked to the nucleic acid cloned in the antisense orientation can be chosen so that the antisense RNA molecule is continuously expressed in a variety of cell types. For example, viral promoters and / or enhancers or regulatory sequences can be chosen so that antisense RNA can be expressed constitutively, organ-specific or cell-type-specific. Antisense expression vectors are recombinant plasmids, phagemids or inactivated viral forms that produce antisense nucleic acids under the control of a highly efficient control region and are active depending on the cell type into which the vector is introduced. Can be determined. Regarding the examination of the control of gene expression using an antisense gene, refer to Weintraub et al. (Review: Trends in Genetics, Volume 1 (No. 1) 1986).

  Another aspect of the invention relates to a host cell into which a recombinant expression vector of the invention is introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms mean not only the cell of interest, but also the progeny or potential progeny of that cell. Because certain modifications occur in generations that are passaged, due to mutations or environmental influences, such progeny may not actually be identical to the parental cells, but still as used herein Included in the scope of terms.

  The host cell can be any prokaryotic or eukaryotic cell. For example, the GAVE6 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (eg, 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 via conventional transformation or transduction techniques. As used herein, the terms “transformation” and “transfection” introduce various foreign nucleic acids (eg, DNA) recognized in the art into host cells. Including co-precipitation with calcium phosphate or calcium chloride, transduction, DEAE-dextran mediated transfection, lipofectin or electroporation.

  For stable transformation of mammalian cells, depending on the expression vector and the transformation technique used, it is known that only a small part of the cell can incorporate foreign DNA into the genome. In order to identify and select integrants, a gene that encodes a selectable marker (eg, antibiotic resistance) is usually introduced into the host cells along with the gene of interest. Preferred selectable markers include those related to resistance to drugs such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding GAVE6 or on a separate vector. Cells that are stably transformed with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated a selectable marker gene can survive even if other cells die). Let ’s)

  A host cell of the invention, eg, a prokaryotic or eukaryotic host cell that has been cultured, can be used to produce (ie, express) GAVE6 protein. Therefore, the present invention further provides a method for producing GAVE6 protein using the host cell of the present invention. In one embodiment, the method comprises the step of culturing the host cell of the invention (introduced with a recombinant expression vector encoding GAVE6) in a culture medium suitable for producing GAVE6 protein. It is. In other embodiments, the method further comprises isolating GAVE6 from the culture medium or the host cell.

In other embodiments, GAVE6 includes an inducible expression system for recombinant expression of other proteins subcloned into a modified expression vector. For example, host cells containing mutant G proteins (eg, yeast cells, Y2 adrenal cells, and cyc ^- S49, US6,168,927B1, 5,739,029 and 5,482,835; Mitchell et al., Proc Natl Acad Sci USA (1992) 89 (19 No.): 8933-37 and Katada et al., J Biol Chem (1984) 259 (6): 3586-95)), such that GAVE6 can be functionally expressed in host cells. An initial expression vector containing a nucleic acid sequence encoding is introduced. Although expressed GAVE6 is constitutively active, the mutation does not lead to signal transduction, and activation of the downstream cascade led by the G protein does not occur (eg, no activation of adenylyl cyclase). Subsequently, a second expression vector is used to transform GAVE6-containing host cells. The second vector carries a structural gene that complements the host protein G protein mutation (ie, functional mammalian or yeast G _s , G _i , G _o , or G _q , eg, WO 97/48820; US 6,168,927 B1, 5,739,029 and 5,482,835), in addition to the genes of interest expressed by the induction system.

  The complementary structural gene of the second vector is inducible; that is, an externally added component that activates a promoter operably linked to the complementary structural gene (eg, tetracycline, IPTG, small molecule Et al., Sambrook et al., Supra). Addition of inducer functionally expresses the protein encoded by the complementary structural gene and constitutively active GAVE6 results in activation of the appropriate downstream pathway (eg, formation of a second messenger) Can be formed. The gene of interest, including the second vector, has a functionally linked promoter that is activated by an appropriate second messenger (eg, CREB, AP1 element). In this way, second messengers accumulate, the promoter upstream of the gene of interest is activated and the product of that gene is expressed. When no inducer is present, the gene expression of interest is switched off.

In certain embodiments, host cells for inducible expression systems include, but are not limited to, S49 (cyc ⁻ ) cells. When a cell line is intended to contain a variant of the G protein, an appropriate variant is artificially created / constructed (see US 6,168,927 B1, 5,739,029 and 5,482,835 for yeast cells).

  In a related aspect, the cell is transformed with a vector operably linked to a cDNA comprising a sequence encoding the protein set forth in SEQ ID NO: 2. First and second vectors containing such systems 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 et al., 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, the host cell may be transformed in any suitable manner where transformation results in functional GAVE6 expression (eg, Sambrook et al., Supra, and Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY, 1990). Such “functional proteins” include, but are not limited to, proteins that are expressed once and form a complex with the G protein that controls the production of second messengers. Other methods applied here for transforming host cells include, but are not limited to, transfection, electroporation, microinjection, transfection ( transduction), cell fusion, DEAE dextran method, calcium phosphate precipitation method, lipofectin (lysosome fusion) method, gene gun method, or DNA vector transporter method (for example, Wu 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, March 15, 1990 application).

  Many types of promoters are applicable in the present invention. Indeed, expression of the polypeptides of the invention may be controlled by any promoter / enhancer known in the art, but such control elements are functional in the host chosen for expression. There must be.

  Promoters that can be used to control GAVE6 expression include, but are not limited to: SV40 early promoter region (Benoist and Chambon, 1981, Nature 290: 304-310), Rous sarcoma virus Promoters containing 3'-long terminal repeats (Yamamoto et al., 1980, Cell 22: 787-797), herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445), regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42); prokaryotic expression vectors such as β-lactamase promoter (Villa-Kamaroff, etc.) 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), or tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25); Furthermore, “Useful Proteins from recombinant bacteria "in Scientific American, 1980, 242: 74-94; yeast or other fungal promoter elements such as the Gal 4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase A promoter; and an animal transcriptional regulatory region that exhibits tissue specificity and has been used in transgenic animals; an elastase I gene regulatory region (active in pancreatic acinar 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 Page); insulin gene regulatory region (active in pancreatic beta cells) (Hanahan, 1985, Nature 315: 115-122), immunoglobulin gene regulatory region (Active in lymphocytes) (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), mouse mammary tumor virus gene regulatory region (active in testis, mammary gland, lymph and mast cells) (Leder et al., 1986, Cell 45: 485-495), albumin gene Regulatory region (active in hepatocytes) (Pinkert et al., 1987, Genes and Devel. 1: 268-276), alphafetoprotein gene regulatory region (active in hepatocytes) (Krumlauf et al., 1985) Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58), alpha 1-antitrypsin gene regulatory region (active in hepatocytes) ( Kelsey et al., 1987, Genes and Devel. 1: 161-171), beta globin gene regulatory region (active in spinal cord cells) (Mogram et al., 198 5 years Nature, 315: 338-340; Kollias et al., 1986, Cell 46: 89-94), myelin base protein gene regulatory region (active in oligodendrocytes of the brain) (Readhead) Et al., 1987, Cell 48: 703-712), myosin light chain-2 gene regulatory region (active in skeletal muscle cells) (Sani, 1985, Nature 314: 283-286), and Gonadotropin-releasing hormone gene regulatory region (active in hypothalamic cells) (Mason et al., 1986, Science 234: 1372-1378).

Expression vectors containing the nucleic acid molecules of the invention can be identified in four general ways: (a) PCR amplification of the desired 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, the nucleic acid is amplified by PCR to provide for detection of the amplified product. In the second method, the presence of an exogenous gene inserted into an expression vector can be detected by nucleic acid hybridization using a probe containing a sequence homologous to the inserted marker gene. In the third method, the recombinant vector / host system is responsible for certain “selectable marker” gene functions (eg, β-galactosidase activity, thymidine kinase activity, resistance to antibiotics, resulting from the insertion of a foreign gene into the vector. Transformation phenotype, presence or absence of baculovirus occlusion body, etc.) can be identified and selected. In another example, when a nucleic acid encoding a GAVE6 protein, variant, analog or derivative thereof is inserted into the “selectable marker” gene sequence of the vector, the recombinant containing the insertion portion is the GAVE6 gene Can be identified by the absence of function. In the fourth method, recombinant expression vectors can be used for the activity, biochemical or immunological activity of the gene product expressed in the recombinant, assuming that the expressed protein has a functional active conformation. Can be identified by assaying the intrinsic properties.

  Many types of host / expression vector combinations can be employed to express the DNA sequences of the present invention. Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, for example. Suitable vectors include derivatives of SV40, known bacterial plasmids such as E. coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67: 31-40 PMB9 and their derivatives, plasmid RP4; phage DNAS, eg, many derivatives of lambda phage (eg, NM989), and other phage DNA (eg, M13 and filamentous single-stranded phage DNA); yeast plasmids (Eg, a 2μ plasmid or derivative thereof; a vector useful in eukaryotic cells (eg, useful in insect or mammalian cells); a vector derived from a combination of plasmid and phage DNA, eg, phage DNA or other expression control sequence; Plasmids modified for use; and the like.

  For example, in baculovirus expression systems, non-fusion transfer vectors such as, but not limited to, pVL941 (BamHl cloning site; Summers), pVL1393 (BamHl, SmaI, XbaI, EcoRl, NotI, XmaIII, BgIII, and PstI cloning sites. Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI, and BamHl cloning sites; Summers and Invitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning sites, blue / white; Invitrogen), and fusion transfer vectors such as, but not limited to, pAc700 (BamHl and KpnI cloning sites, where the BamHl recognition site begins with the start codon; Summers), pAc701 And pAc702 (same reading frame as pAc700), pAc360 (BamHl cloning site, polyhydrin open 36 base pairs downstream of the start codon; Invitrogen (195)), and pBlueBacHisA, B, C (three different reading frames, BamH1, BglII, PstI, NcoI, and HindIII cloning sites, for ProBond purification Screening recombinant plaques with N-terminal peptides and blue / white; Invitrogen (220)) can be used.

  Mammalian expression vectors contemplated for use in the present invention include vectors having inducible promoters, such as all expression vectors having a dehydrofolate reductase (DHFR) promoter (eg, DHFR expression vector). ), Or DHFR / methotrexate co-amplification vectors, eg, pED (expressing both cloned genes and DHFR at the PstI, SalI, SbaI, SmaI, and EcoRI cloning sites; Kaufman, Current Protocols in Molecular Biology, 16.12 (1991 ) See)). Instead, a glutamine synthetase / methionine sulfoximine co-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning sites, where the vector expresses glutamine synthetase and the cloned gene; Celltech). In other embodiments, vectors that direct episomal expression under the control of Epstein Barr virus (EBV) can be used, and examples of such vectors include pREP4 (BamHl, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning sites, constitutive RSV-LTR promoter, hygromycin selectable marker; Invitrogen, pCEP4 (BamHl, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen, pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamHl cloning site, inducible metallothionein Iia Gene promoter, hygromycin selectable marker; Invitrogen), pREP8 (BamHl, XhoI, NotI, HindIII, NheI, and KpnI cloning Site, 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 can be purified with ProBond resin and then cleaved with enterokinase; Invitrogen). Selectable mammalian expression vectors used in the present invention include pRc / CMV (HindIII, BstXI, NotI, SbaI, and ApaI cloning sites, G418 selection; Invitrogen), pRc / RSV (HindIlI, SpeI, BstXI , NotI, XbaI cloning site, G418 selectable marker; Invitrogen), and others. Vaccinia viral mammalian expression vectors (Kaufman, 1991, supra) for use in the present invention include, but are not limited to, pSC11 (SmaI cloning site, TK- and β-gal selection), pMJ601 (SalI, SmaI, AfII, NarI, BspMII, BamHI, ApaI, NheI, SacII, KpnI, and HindIII cloning sites, TK- and β-gal selection) and pTKgptF1S (EcoRI, PstI, SalI, Accl, HindII, SbaI , BamHI, and Hpa cloning sites, TK or XPRT selection).

  In addition, yeast expression systems can be used according to the present invention to express GAVE6 protein, variants, analogs or derivatives thereof, examples of which include two non-fusion types: pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamHl, SacI, Kpnl, and HindIII cloning sites; Invitrogen) or fused pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI EcoRI, BamHl, SacI, KpnI, and HindIII cloning sites, N-terminal peptides that can be purified with ProBond resin and then cleaved with enterokinase (Invitrogen) can be employed in the present invention.

  Once a particular recombinant DNA molecule has been identified and isolated, it can be propagated in a number of ways known in the art. Once the appropriate host system and culture conditions are established, the recombinant expression vector can be propagated and a certain amount can be prepared. As noted above, expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses (eg, vaccinia virus or adenovirus; insect viruses (eg, baculovirus); Yeast vectors; bacteriophage vectors (eg, lambda), and plasmid and cosmid DNA vectors, to name a few.

  In addition, the host cell strain can be selected from those that modulate the expression of the inserted sequences or those in which the gene product is modified or processed in the specific fashion desired. Different host cells have characteristic and specific mechanisms for translation and post-translational processing as well as protein modifications (eg, glycosylation, cleavage [eg, signal sequences]). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to obtain a non-glycosylated core protein product.

Transgenic animals The host cells of the invention can also be used to create non-human transgenic animals. For example, in one embodiment, the host cell of the present invention is a fertilized egg or embryonic stem cell into which a sequence encoding GAVE6 has been introduced. Such host cells can then be used to create non-human transgenic animals in which the exogenous GAVE6 sequence has been integrated into the genome, or homologous recombinant animals in which the endogenous GAVE6 sequence has been modified. can do. Such animals are useful for studying the function and / or activity of GAVE6 and for identifying and / or evaluating modulators of GAVE6 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 are present. It contains a transgene. Examples of other 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 a cell from which a transgenic animal grows and is retained in the genome of the grown animal. This transgene directs the expression of the encoded gene product in the tissues of the transgenic animal or in one or more cell types. As used herein, “homologous recombinant animal” means a non-human animal, preferably a mammal, more preferably a mouse, whose endogenous GAVE6 gene has been modified by homologous recombination. To do. This recombination is carried out between the endogenous GAVE6 gene and an exogenous DNA molecule introduced into the animal cell, for example, the embryonic cell of the animal before growing into the animal.

  The transgenic animal of the present invention can be prepared by introducing a nucleic acid molecule encoding GAVE6 into the male pronuclei of a fertilized egg cell by one of the transformation methods described above. The egg cell is then developed in a pseudopregnant female foster animal. A GAVE6 cDNA sequence, such as that of SEQ ID NO: 1, can be introduced into the genome of a non-human animal, for example, as a transgene. Alternatively, a non-human homologue of the human GAVE6 gene, such as the mouse GAVE6 gene, can be isolated based on hybridization to human GAVE6 cDNA and can be used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. Tissue specific regulatory sequences are operably linked to the GAVE6 transgene and direct the expression of the GAVE6 protein in specific cells. Methods for generating transgenic animals by embryo manipulation and microinjection are routinely used in the art, particularly in animals such as mice, for example, US 4,736,866 and 4,870,009, US 4,873,191. And Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). Similar methods can be used to create other transgenic animals with the transgene in the genome and / or to express GAVE6 mRNA in the cells or tissues of the animal. The transgenic animals can then be used to further breed animals carrying the transgene. In addition, a transgenic animal carrying a transgene encoding GAVE6 can be further mated with a transgenic animal carrying another transgene.

  To create a homologous recombinant animal, the vector is at least a portion of a GAVE6 gene (eg, a human or non-human homologue of the GAVE6 gene, eg, a rodent GAVE6 gene), thereby deleting, adding or replacing Are introduced for modification, for example, to include those in which the function of the GAVE6 gene is disrupted. In certain embodiments, the vector can be designed such that, in homologous recombination, the endogenous GAVE6 gene is functionally disrupted (ie, no longer encodes a functional protein; a “knockout” vector. Also called).

  Instead, the vector encodes a protein that is still functional in homologous recombination while the endogenous GAVE6 gene is mutated or otherwise altered (eg, upstream of the control region thereby endogenously). Can be modified to alter the expression of the sex GAVE6 protein.

  In the homologous recombination vector, the modified segment of the GAVE6 gene is bordered by additional nucleic acid sequences of the GAVE6 gene at its 5 ′ and 3 ′ ends, and in fetal stem cells, the exogenous GAVE6 gene carried by the vector and Homologous recombination occurs between the endogenous GAVE6 genes. This added GAVE6 flanking nucleic acid sequence is of sufficient length to allow successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both 5 ′ and 3 ′ ends) are included in the vector (eg, Thomas et al., Cell (1987) 51: 503). (See description of recombinant vector).

  This vector is introduced into fetal stem cells (eg, by electroporation) and cells in which the introduced GAVE6 gene is recombining with the endogenous GAVE6 gene are selected (eg, Li et al., Cell (1992) 69: 915). The selected cells are injected into an animal (eg, mouse) blastocyst to form a chimeric pseudo-aggregate (eg, Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, edited by Robertson, IRL, Oxford, (1987) pp. 113-152). The chimeric embryo is then transferred to an appropriate pseudopregnant foster animal, and the embryo is maintained in a pregnant state until birth. Progeny animals bearing homologous recombinant DNA in germ cells can be used to breed animals in which all animal cells contain the homologous recombinant DNA by propagation of the transgene into the germline.

  Methods for producing homologous recombination vectors and homologous recombination animals are further described in Bradley, Current Opinion in Bio / Technology (1991) 2: 823-829 and WO90 / 11354, WO91 / 01140, WO92 / 0968 and WO93 / 04169.

  In other embodiments, the transgenic non-human animal can be engineered to include a selection system that can control the expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage PI. For a description of this cre / loxP recombinase system, see, for example, Lakso et al., Proc Natl Acad Sci USA (1992) 89: 6232-6236. An example of another recombinase system is the S. cerevisiae FLP recombinase system (O'Gorrnan et al., Science (1991) 251: 1351-1355). If the cre / loxP recombinase system is used to control transgene expression, an animal containing a transgene encoding both the cre recombinase and the selected protein is required. Such animals can be produced by creating “double” transgenic animals, for example in two transgenic animals, one containing a transgene encoding the selected protein and the other a transgene encoding a recombinase. Provided by mating animals containing the gene.

  The non-human transgenic animal clones described herein can also be made by the methods described in Wilmut et al., Nature (1997) 385: 810-813 and WO97 / 07668 and WO97 / 07669. Briefly, cells from transgenic animals, eg, somatic cells, can be isolated and allowed to exit the growth cycle and enter the Go phase. The stationary phase cells can then be fused with enucleated egg cells obtained from the same species of animal from which the stationary phase cells were obtained, eg, by use of electric pulses. The reconstructed egg cells are then cultured until they become morulas or blastocysts and transplanted into pseudopregnant female foster animals. A pregnant offspring of a female foster animal will be a clone of the animal from which its cells, eg, somatic cells, have been isolated.

Pharmaceutical Compositions GAVE6 nucleic acid molecules, GAVE6 proteins and anti-GAVE6 antibodies (also referred to herein as “active compounds”) of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise a nucleic acid molecule, protein or antibody, and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, diffusing agents, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents that are compatible with pharmaceutical administration. , And their analogs. Such carriers and agents for pharmaceutically active substances are well known in the art. A commonly used carrier or drug is intended for use in the composition unless it is incompatible with the active compound. Additional active compounds can also be combined with the composition.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral routes such as intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration can contain the following components: water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, polyethylene glycol, or other Aseptic diluents such as solvents: antibacterial agents such as benzyl alcohol or methylparaben: antioxidants such as ascorbic acid or sodium bisulfite: chelating agents such as EDRA: acetate, citrate or phosphate And isotonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as HCl or NaOH. The parenteral preparation can also be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

  Pharmaceutical compositions suitable for injectable administration include sterile aqueous solutions (water-miscible) or diffusion solutions and sterile powders for the preparation of sterile injectable solutions or diffusion solutions. For intravenous injection, suitable carriers include physiological saline, sterile water, “CREMOPHOR EL” (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solution or diffusion liquid, and includes, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersion and by the use of surfactants. Protection against the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an absorption delaying agent, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions may contain the active compound (eg, GAVE6 protein, variant thereof, or analog or derivative thereof; or anti-GAVE6 antibody) in the required amount, in an appropriate solvent, as necessary, as listed above. One or a combination of the ingredients can be incorporated and then sterile filtered. In general, diffusion solutions can be prepared by incorporating the active compound into a sterile carrier that contains a basic diffusing agent and any of the ingredients listed above. In the case of sterile powders for the preparation of sterile injection solutions, the preferred method of manufacture consists of the active sterile powder and any additional ingredients desired by vacuum drying and lyophilization from a previously sterile filtered solution. To get a good powder.

  Oral compositions generally include an inert diluent or an edible carrier. The composition is enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound is mixed with excipients and used in the form of tablets, troches, or capsules. Oral compositions are also manufactured using a liquid carrier for use as an gargle, where the active compound is administered orally and swished, expired or swallowed You can also

  Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; like starch or lactose Excipients; disintegrants such as alginic acid, Primogel or corn starch; lubricants such as magnesium stearate or Sterotes; brighteners such as colloidal silicon dioxide; such as sucrose or saccharin Sweeteners; flavor correctors such as peppermint, methyl salicylate or orange flavors. For inhalation administration, the compounds are released in the form of an aerosol spray or nebulizer from a pressurized container or dispenser containing a suitable high pressure gas, such as carbon dioxide.

  Systemic administration is also performed by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, surfactants, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels, or creams as generally known in the art.

  The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal release.

  In certain embodiments, the active compounds can be prepared as controlled drug release dosage forms, including implants and microencapsulated release systems, with carriers that can protect the compound against rapid elimination from the body. . Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

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

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of administration. As used herein, a dosage unit form is a physically distinct unit suitable for integrated dosing for the patient being treated; each unit together with the required pharmaceutical carrier Contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg, 0.1 to 20 mg / kg) of compound may be administered to the patient, eg, by one or more doses or continuous infusions. Candidate dose at start of administration. A typical daily dose is about 1 μg / kg to 100 mg / kg or more, depending on the factors described above. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until a desired symptom suppression occurs. However, other modes of administration are useful. The progress of treatment is easily monitored by conventional techniques and measurement methods. An exemplary mode of administration is disclosed in WO 94/04188. The specifications for the dosage unit forms of the present invention are determined by the unique nature of the active compound and the particular therapeutic effect to be achieved and the limitations inherent in the art of the formulation of the active compound for the treatment of individuals. And is directly dependent.

  Furthermore, the nucleic acid molecule of the present invention can be incorporated into a vector and used as a gene therapy vector. Gene therapy vectors are described, for example, by intravenous injection, local administration (US 5,328,470) or stereotactic injection (eg, Chen et al., Proc Natl Acad Sci USA (1994) 91: 3054-3057, see It can be applied to patients. The gene therapy vector pharmaceutical preparation can include the gene therapy vector in an acceptable diluent, or can include a delayed release matrix in which the gene delivery vehicle is embedded. Alternatively, when a complete gene delivery vector can be produced intact from a recombinant cell (eg, a retroviral vector), the pharmaceutical preparation can include one or more cells capable of producing a gene delivery system. it can.

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

Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues and antibodies of the invention, and fragments thereof can be used in one or more of the following methods: a) screening assays; b) detection assays ( C) predictive medicine (eg, diagnostic assays, prognostic assays, clinical trial monitoring and pharmacogenomics); and d) therapeutic methods (eg, therapeutic and Prophylactic). The GAVE6 protein interacts with other intracellular proteins and can then be used for (i) control of cell proliferation; (ii) control of cell differentiation; and (iii) control of cell survival. The isolated nucleic acid molecules of the invention can be used to express GAVE6 protein (eg, via a recombinant expression vector in a host cell for gene therapy applications), or to detect GAVE6 mRNA (eg, in a biological sample). ), Or to detect GAVE6 gene damage and to modulate GAVE6 activity. Furthermore, GAVE6 protein can be used to screen for drugs or compounds that modulate GAVE6 activity or expression, and for the treatment of diseases characterized by insufficient or excessive production of GAVE6 protein. Screening for the production of GAVE6 protein with reduced or abnormal activity compared to GAVE6 native protein can also be achieved by the present invention. Furthermore, the anti-GAVE6 antibodies of the present invention can be used for detection and isolation of GAVE6 protein and for modulation of GAVE6 activity. The present invention further relates to novel agents identified by the screening assays described above and their therapeutic uses as described herein.

Screening assay In the presence of endogenous ligand, activation of the G protein receptor leads to the formation of a G protein receptor complex, which leads to the binding of GTP to the G protein. The GTPase domain of the G protein slowly hydrolyzes GTP to GDP, resulting in receptor inactivation under normal conditions. However, in the presence of a constitutively activated receptor, hydrolysis of GTP to GDP continues.

A non-hydrolyzable substrate for G protein, [ ³⁵ S] GTPγS, can be used to monitor facilitated binding to membranes expressing constitutively activated receptors. Traynor and Nahorski report that [ ³⁵ S] GTPγS can be used to monitor the coupling of G proteins to membranes in the presence and absence of ligand (Traynor et al., Mol Pharmacol (1995). 47 (4): 848-54). The use of such preferred assay systems is generally applicable to all G protein-coupled receptors without regard to the specific G protein that binds to that receptor, so that for initial screening of candidate compounds. Is the use of.

G _s20 is the enzyme adenylyl cyclase activation, whereas G _i and G _o inhibit the enzyme. As is well known in the art, adenylyl cyclase catalyzes the conversion of ATP to cAMP. Thus, constitutively activated GPCRs coupled to G _s proteins are associated with increased intracellular concentrations of cAMP. Instead, constitutively activated GCPR, which may be coupled to G _i (or G _o ) protein, is associated with a reduced intracellular concentration of cAMP. See "Indirect Mechanism of Synaptic Transmission", Chapter 8, From Neuron to Brain (3rd edition), Nichols et al., Sinauer Associates, Inc., 1992. Thus, assays that detect cAMP can be used to determine whether a candidate compound is an inverse agonist to the receptor. Methods known in the art can be used for various cAMP measurements. In one embodiment, anti-cAMP antibodies can be used in an ELISA-based format. In other embodiments, a whole cell second messenger reporter system assay is contemplated (see WO00 / 22131).

  In a related aspect, cyclic AMP is expressed through the promotion of binding of a cAMP-reactive DNA binding protein or a transcription factor (CREB) that binds to a promoter at a specific site called a cAMP response element. Promote. Thus, a reporter system can be constructed that has a promoter that includes multiple cAMP response elements in front of a reporter gene (eg, β-galactosidase or luciferase). Furthermore, the constitutively activated Gs-coupled receptor causes cAMP accumulation, which in turn activates the gene and expresses the reporter protein. The reporter protein (eg, β-galactosidase or luciferase) can then be detected by standard biochemical assays (see WO00 / 22131).

Other G proteins, e.g., G _o and G _q, enzymes are associated with the activation of phospholipase C, followed by phospholipid, PIP2, hydrolyzing a two intracellular messengers, diacylglycerol (DAG) And inositol 1,4,5-triphosphate (IP3). Accumulation of increased IP3 is associated with activation of the G _q-coupled receptor and G _o-coupled receptors (see WO00 / 22131). Assays that detect IP3 accumulation can be used to a candidate compound to determine whether the inverse agonist for G _q-coupled receptor or G _o-coupled receptor. G _q -coupled receptors can also be examined using an AP1 reporter assay that measures whether G _q- dependent phospholipase C causes activation of a gene containing an AP1 element. Thus, activated G _q-coupled receptor, it can prove to be increased in the expression of such genes, whereby it will be demonstrated that inverse agonist has reduced the such expression.

  Here also candidate compounds or test compounds or agents (for example peptides, peptidomimetics, which bind to modulators, i.e. GAVE6 protein or have a stimulatory or inhibitory effect on GAVE6 expression or GAVE6 activity, for example). Methods for identifying small molecules, or other pharmaceutical agents (also referred to herein as “screening assays”) are provided.

  In one embodiment, the invention provides an assay for screening candidate or test compounds that binds to or modulates membrane-bound GAVE6 protein, polypeptide or portion thereof having biological activity. Is provided. The test compounds of the present invention can be obtained using any of a vast number of approaches in combinatorial library methods known in the art, such libraries include: A spatially addressable parallel solid or liquid phase library; a synthetic library method that requires deconvolution; a “one bead one compound” library; and an affinity chromatography selection Synthetic library method using the method. Biological library approaches are limited to peptide libraries, and the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers, or compounds (Lam, Anticancer Drug Des (1997). ) 12: 145).

  Examples of methods for synthesizing molecular libraries can be found in the art, for example: DeWitt et al., Proc Natl Acad Sci USA (1993) 90: 6909; Erb et al., Proc Natl Acad Sci 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., J Med Chem (1994) 37: 1233.

  A library of compounds can exist in solution (eg, HoughtenBio / Techniques (1992) 13: 412-421) or on beads (Lam, Nature (1991) 354 : 82-84), chip (Fodor, Nature (1993) 364: 555-556), bacteria (US 5,223,409), spore (US 5,571,698; 5,403,484; and 5,223,409), plasmid ( Cull et al., Proc Natl Acad Sci USA (1992) 89: 1865-1869) or even phage (Scott et al., Science (1990) 249: 386-390; Devlin, Science (1990) 249 Volume: 404-406; Cwirla et al., Proc Natl Acad Sci USA (1990) 87: 6378-6382; and Felici, J Mol Biol (1991) 222: 301-310).

A particular embodiment of the present invention is a cell-based assay, wherein the cell expresses the GAVE6 protein or biologically active portion thereof on the cell surface in a membrane-bound manner, Contact with a test compound and measure the ability of the test compound to bind to the GAVE6 protein. The cell is, for example, a yeast cell or a mammal-derived cell. Measurement of the binding ability between the test compound and the GAVE6 protein can be accomplished, for example, by labeling the test compound with a radioisotope or enzyme and binding the test compound to the GAVE6 protein or biologically active portion thereof as a complex. It can be carried out by detecting and measuring the labeled compound. For example, test compounds are labeled directly or indirectly with ¹²⁵ I, ³⁵ S, ¹⁴ C or ³ H, and radioisotopes are detected by direct counting of radiation emission or scintillation measurements. Alternatively, the test compound can be enzymatically labeled, for example the enzyme can be horseradish peroxidase, alkaline phosphatase or luciferase, and the enzyme label is detected by measuring the conversion of the appropriate substrate. In certain embodiments, the assay comprises contacting a cell wherein a GAVE6 protein or biologically active portion of its membrane-bound form is expressed on the cell surface with a compound known to bind to GAVE6. And then the assay mixture is contacted with the test compound and the ability of the test compound to interact with the GAVE6 protein is determined, wherein the test compound's ability to interact with the GAVE6 protein is determined by determining whether the test compound is a GAVE6 protein Or measuring the ability of the biologically active portion to bind preferentially compared to a known compound.

In another embodiment, the assay is a cell-based assay, wherein a cell expressing a GAVE6 protein or a biologically active portion of the membrane-bound form thereof on the cell surface is contacted with a test compound, Measuring the ability to modulate (eg, promote or inhibit) the activity of a GAVE6 protein or biologically active portion thereof. The ability of a test compound to modulate the activity of a GAVE6 protein or biologically active portion thereof is achieved, for example, by measuring the ability of the GAVE6 protein to bind to or interact with a GAVE6 target molecule. Can do. As used herein, a “target molecule” is one that naturally binds or interacts with a GAVE6 protein, for example, a cell surface molecule that expresses the GAVE6 protein, a second cell surface molecule, There are molecules in the extracellular environment, molecules associated with the inner surface of the cell membrane, or intracellular molecules. The GAVE6 target molecule can be a non-GAVE6 molecule or a GAVE6 protein or polypeptide of the invention. In one embodiment, the GAVE6 target molecule is a component of a signal transduction pathway, and an extracellular signal (eg, a signal generated by the binding of a membrane-bound GAVE6 molecule and a compound) into the cell through the cell membrane. It is intended to promote communication. The target can be, for example, a secondary intracellular protein with catalytic activity or a protein that, together with GAVE6, promotes association with downstream signaling molecules.

Measuring the ability of a GAVE6 protein to bind to or interact with a GAVE6 target molecule can be accomplished using one of the methods for measuring direct binding described above. In certain embodiments, measuring the ability of a GAVE6 protein to bind or interact with a GAVE6 target molecule can be accomplished by measuring the activity of the target molecule. For example, the activity of the target molecule can be detected by detecting second-order messenger induction of the target (eg, intracellular Ca ²⁺ , diacylglycerol, IP3, etc.), catalytic / enzymatic activity of the target on the appropriate substrate. Detection of reporter genes (eg, GAVE6 responsive regulatory elements operably linked to nucleic acids encoding markers for detection such as luciferase) or cell responses, eg, cell differentiation or cell It can be measured by detecting growth.

  The invention further comprises contacting the GAVE6 protein or biologically active portion thereof with a test compound and measuring the ability of the test compound to bind to the GAVE6 protein or biologically active portion thereof. Cell-free assays, including Binding of the test compound to the GAVE6 protein can be measured directly or indirectly as described above. In a preferred embodiment, the assay is contacted with a GAVE6 protein or biologically active portion thereof with a compound that binds to a known GAVE6 to form an assay mixture, which is then contacted with the test compound. Measuring the ability of a test compound to interact with the GAVE6 protein, wherein determination of the ability of the test compound to interact with the GAVE6 protein is determined by determining whether the test compound is a GAVE6 protein or a biologically active portion thereof. And measuring the ability to bind preferentially over known compounds.

  Other cell-free assays of the invention contact the GAVE6 protein or biologically active portion thereof with a test compound, and then the test compound determines the activity of the GAVE6 protein or biologically active portion thereof. Measuring the ability to modulate (eg, promote or inhibit). Determination of the ability of a test compound to modulate GAVE6 activity can be accomplished, for example, by measuring the ability of a GAVE6 protein to bind to a GAVE6 target molecule using one of the methods described above that directly measure binding. it can. In other embodiments, the determination of the ability of a test compound to modulate GAVE6 activity is determined by the ability of the GAVE6 protein to further modulate the GAVE6 target molecule, eg, catalytic / to the appropriate substrate of the target molecule that can be measured as described above. It can be achieved by measuring enzymatic activity.

In addition, other cell-free assays of the invention may be made by contacting a GAVE6 protein or biologically active portion thereof with a known GAVE6 binding compound to form an assay mixture, wherein the assay mixture and test compound are contacted. Contacting and then measuring the ability of the test compound to interact with the GAVE6 protein. Measuring the ability of a test compound to interact with the GAVE6 protein includes measuring the ability of the GAVE6 protein to preferentially bind or modulate a GAVE6 target molecule.

Receptor is a non-ligand molecule, which does not necessarily inhibit ligand binding, but is a receptor that binds to the G protein or possibly condenses, dimerizes, or clusters and activates the receptor It is activated by those that cause structural changes of For example, antibodies are raised against various portions of GAVE6 exposed on the cell surface. These antibodies activate cells as measured by standard assays (eg, monitoring cAMP concentration or intracellular Ca ²⁺ concentration) via the G protein cascade. Monoclonal antibodies may be preferred because they include molecular mapping, and in particular epitope mapping. Monoclonal antibodies can be generated against intact receptors expressed on the cell surface and peptides known to be formed on the cell surface. In order to obtain a large number of related peptides, the method of Geysen et al., US 5,998,577 can be performed. Antibodies found to activate GAVE6 are modified to minimize activities unrelated to GAVE6 activation, such as complement fixation. Thus, the antibody molecule is shortened or mutated to minimize or eliminate activities other than GAVE6 activation. For example, for certain antibodies, only an antigen binding site is required. Thus, the _Fc region of the antibody is removed.

  Cells expressing GAVE6 are exposed to the antibody and activate GAVE6. Activated cells are then exposed to various molecules to identify which molecules modulate receptor activity, resulting in higher or lower activation levels. Molecules that achieve such a purpose are then subjected to cells expressing GAVE6 without antibodies and tested to observe the effect on non-activated cells. This target molecule can then be tested and modified as a candidate drug for the treatment of diseases associated with altered GAVE6 metabolism using known techniques.

In the cell-free assay of the present invention, both lytic and membrane-bound GAVE6 can be used. In the case of a cell-free assay containing membrane-bound GAVE6, it is desirable to use a solubilizing agent so that membrane-bound GAVE6 can be retained in the solution. Examples of such solubilizers include n-octyl glycoside, n-dodecyl glycoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, TRITON X-100, TRITON X-114, THESIT, isotridecyl poly (ethylene glycol ether) _n , 3-[(3-chloroamidopropyl) dimethylammino] -1-propanesulfonate (CHAPS), 3-[(3-chloroamidopropyl) dimethylammino] Nonionic surfactants such as -2-hydroxy-1-propanesulfonate (CHAPSO) or N-dodecyl = N, N-dimethyl-3-ammonio-1-propanesulfonate are included.

  Furthermore, in one or more of the above assay method embodiments of the invention, immobilizing either GAVE6 or its target molecule to facilitate separation of the complex from uncomplexed one or both proteins. At the same time, it is desirable to support automation of the assay.

Binding of the test compound to GAVE6 or interaction with the target molecule of GAVE6 in the presence or absence of the candidate compound can be achieved in any container suitable for containing the reactants. Examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein is provided that adds a domain so that one or both proteins can be bound to a matrix. For example, glutathione-S-transferase / GAVE6 fusion protein or glutathione-S-transferase / target fusion protein can be adsorbed to glutathione SEPHAROSE beads (Sigma Chemical, St. Louis, MO).

  Instead, the glutathione derivatized microtiter plate is then conjugated to or a test compound. Subsequently, the mixture with either the non-adsorbed target protein or the GAVE6 protein is allowed to incubate under conditions that allow complex formation (eg, under physiological salt and pH conditions). Following the reaction, the beads or microtiter plate are washed to remove unbound components and the presence of complex formation is measured either directly or indirectly. Alternatively, the complex is separated from the matrix and the level or activity of GAVE6 binding can be measured by standard techniques.

  Other techniques for immobilizing proteins on a matrix can also be used in the screening assays of the invention. For example, GAVE6 or any of its target molecules can be immobilized using conjugation with biotin and streptavidin. Biotinylated GAVE6 or target molecule can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, IL) , Immobilized on the wall of a 96-well plate (Pierce Chemicals) coated with streptavidin. Alternatively, antibodies that react with GAVE6 or a target molecule but do not interfere with the binding of the GAVE6 protein to the target molecule can be derivatized to the wall of the plate. In the reaction, unbound target or GAVE6 is trapped on the wall by the antibody conjugate. Methods for detecting such complexes include, in addition to those described above for GST-immobilized complexes, immunological detection of complexes using antibodies that react with GAVE6 or target molecules, and GAVE6 or target molecules Also included are enzyme binding assays based on detecting the relevant enzyme activity.

  In other embodiments, modulators of GAVE6 expression are identified by a method of contacting a cell with a candidate compound and measuring the expression of GAVE6 mRNA or protein in the cell. In the presence of the candidate compound, the expression level of GAVE6 mRNA or protein is compared to the expression level of GAVE6 mRNA or protein in the absence of the candidate compound. Candidate compounds can then be identified as modulators of GAVE6 expression based on the comparison. For example, in the presence of a candidate compound, when the expression of GAVE6 mRNA or protein is greater than in its absence (statistically significantly greater), the candidate compound is a promoter or agonist of GAVE6 mRNA or protein expression Identified. Alternatively, in the presence of a candidate compound, when the expression of GAVE6 mRNA or protein becomes smaller (statistically significantly smaller) in the absence, the candidate compound is an inhibitor or an inhibitor of GAVE6 mRNA or protein expression Identified as an antagonist. If GAVE6 activity is reduced below baseline in the presence of a ligand or agonist, or in constitutive GAVE6, the candidate compound is identified as an inverse agonist. The intracellular level of GAVE6 mRNA or protein expression can be measured by the methods described herein for detection of GAVE6 mRNA or protein.

  In yet another aspect of the invention, the GAVE6 protein is used as a “bait protein” in a two-hybrid assay or a three-hybrid assay (US 5,283,317; Zervo et al., Cell (1993) 72: 223-232; Madura et al., J Biol Chem (1993) 268: 12046-12054; Bartel et al., Bio / Techniques (1993) 14: 920-924; Iwabuchi et al. Oncogene (1993) 8: 1693-1696; and WO94 / 10300; see) other proteins that bind to or interact with GAVE6 and modulate GAVE6 activity ("GAVE6 binding protein" or "GAVE6- bp ") can be identified. Such GAVE6 binding proteins may be involved in signal transduction by the GAVE6 protein, for example, upstream or downstream elements of the GAVE6 pathway.

  The present invention allows large-scale production of purified GAVE6 and allows the physical properties of the conformation of the region that would have a function to be analyzed for rational drug design. For example, the IC3 region and EC domain of the molecule are regions of particular interest. Once the morphology of the region and the composition of ions are known, candidate drugs for interacting with such regions can be established and then tested in intact cells, animals and patients. Such methods capable of extracting three-dimensional structure information include X-ray crystallography, NMR spectroscopy, molecular modeling, and the like. The three-dimensional structure can also lead to the identification of similar conformational sites in other known proteins for which there are known pharmaceutical agents acting at that site. These pharmaceuticals or their derivatives can be found using GAVE6.

  The present invention further relates to novel pharmaceutical agents identified by the screening assays described above, and their use for therapy as described herein.

Assay of the present invention
A. Detection Assays A portion or fragment of the DNA sequence of the present invention can be used in many ways as a polynucleotide reagent. For example, the sequence includes (i) a map of each gene in the chromosome and the location of the genetic region associated with the genetic disease; (ii) identification of individuals from small biological samples (tissue typing) And (iii) can be used to aid in forensic identification of biological samples. These applications are described in the following subsections.

1. Chromosome mapping Once the sequence of a gene (or a portion of the sequence) has been isolated, the sequence can be used to map the location of the GAVE6 gene on the chromosome. The GAVE6 nucleic acid molecules described herein or fragments thereof can be used to map the location of the GAVE6 gene on the genome. Mapping the location of the GAVE6 sequence in the genome (especially the human genome) is an important first step in correlating the gene and its sequence associated with disease.

  Briefly, the GAVE6 gene can be mapped in the genome by creating PCR primers (preferably 15-25 bp long) from the GAVE6 sequence. This primer is used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only hybrids containing the human gene corresponding to the GAVE6 sequence form an amplified fragment.

  Somatic cell hybrids are prepared by fusing somatic cells obtained from different mammals (eg, human and mouse cells). When a hybrid of human and mouse cells grows and divides, generally human chromosomes are randomly lost, but mouse chromosomes are retained. Using a medium in which mouse cells cannot grow (due to specific enzyme deficiencies) but human cells can grow, one human chromosome containing the gene encoding the required enzyme will be retained. A panel of hybrid cell lines was established using various media. Each cell line in the panel contains either a single human chromosome or a small number of human chromosomes, and the entire set of mouse chromosomes, making it easy to map individual genes to specific human chromosomes (D'Eustachio et al., Science (1983) 220: 919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced using human chromosomes with translocations and deletions.

  PCR mapping with somatic cell hybrids is a rapid method for assigning specific sequences to specific chromosomes. Three or more sequences per day can be allocated using a single thermocycler.

  Similarly, other mapping strategies that can be used to map GAVE6 sequences to specific chromosomes in the genome are in situ hybridization (Fan et al., Proc Natl Acad Sci USA (1990) 87: 6223-27. Page), pre-screening using flow-sorted labeled chromosomes, and pre-selection by hybridization to a chromosome-specific cDNA library.

  Fluorescence in situ hybridization (FISH) of DNA sequences to metaphase chromosomal spread can also be used to provide precise chromosomal location in one step. Stretched chromosomes can be created using cells whose cell division is in mid-mitosis and blocked by a chemical that destroys the spindle (eg, colcemid). In simple terms, the chromosomes are first treated with trypsin and then stained with Giemsa. Light and dark bands are developed on each chromosome to identify individual chromosomes. FISH technology can be performed using short DNA sequences of 500 to 600 bases. However, clones larger than 1000 bases are more likely to bind to a single chromosome location with sufficient light intensity with only one detection. Preferably 1000 bases, and more preferably 2000 bases are sufficient to give good results within a reasonable time. For a review of this technique, see Verma et al. (Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988)). Chromosome mapping can be inferred in silico and statistical considerations such as rod scores or simply proximity can be employed.

  Chromosomal mapping reagents can be used to individually locate a single site on a chromosome. In addition, a panel of reagents can be used to mark multiple locations and / or multiple chromosomes. Reagents corresponding to the flanking sequences of the GAVE6 gene are actually preferred for mapping purposes. Coding sequences are likely conserved within gene families, thereby increasing the chance of cross-hybridization during chromosomal mapping.

  Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data (such as McKusick, Mendelian Inheritance in Man, Available online via Johns Hopkins University, Welch Medical Library). The relationship between genes and diseases mapped to the same chromosomal region can be identified by linkage analysis (co-inheritance of physically adjacent genes), eg, Egeland et al., Nature ( 1987) 325: 783-787.

In addition, DNA sequence differences between individuals with and without a disease associated with GAVE6 are determined. If the mutation is observed in individuals with some or all diseases but not in all unaffected individuals, the mutation may be the etiology responsible for the particular disease Is expensive. Comparison of affected and unaffected individuals is generally the first structural change in the chromosome, such as a deletion or translocation, which can be performed either visually from the chromosomal spread or by PCR based on the DNA sequence. And can be detected. Ultimately, complete sequencing of genes from various individuals is performed to determine the presence of the mutation and to distinguish the mutation from polymorphism.

2. Tissue typing
The GAVE6 sequences of the present invention can be used to identify individuals from small biological samples. For example, the US Army is considering using restriction enzyme fragment length polymorphism (RFLP) to identify individuals. In this technique, an individual's genomic DNA is cut with one or more restriction enzymes and confirmed by Southern blotting to produce a unique band for identification. This method relieves the limitations of the currently used “Dog Tags”, that is, disappearance, replacement, and theft, making it difficult to identify clearly. The sequences of the present invention are useful as additional DNA markers for RFLP (described in US 5,272,057).

  In addition, the sequences of the present invention can be used to provide an alternative method for determining selected segments of an individual's genome with actual base-to-base DNA sequences. Thus, the GAVE6 sequence described here can be used to prepare two PCR primers from the 5 ′ and 3 ′ sides of the sequence. This primer can then be used to amplify the individual's DNA and subsequently provide its sequence.

  The panel of corresponding DNA sequences derived from individuals prepared in this way is a method of unique personal identification (ie because each individual has a unique set of such DNA sequences due to allelic differences) I will provide a. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissues. The GAVE6 sequence of the present invention uniquely represents a portion of the human genome. Allelic variation occurs at a certain frequency in the coding region of the sequence and is frequent in the non-coding region. Allelic variation between human individuals occurs at a rate of about every 500 bases. Each of the sequences described herein can be used as a standard against which, to some extent, DNA from individuals can be compared for personal identification purposes. Because there are more genetic polymorphisms in the non-coding region, fewer sequences are needed to identify the individual. The non-coding sequence of SEQ ID NO: 1 can provide effective personal identification with a panel of perhaps 10 to 1000 primers, each producing a non-coding amplified sequence of 100 bases. If the predicted coding sequence (eg, SEQ ID NO: 1) was used, a more appropriate number of primers for effective personal identification would be 500 to 2000.

  If the panel of reagents from the GAVE6 sequence described here was used to create a unique identification database for an individual, those same reagents could later be used to identify tissue from that individual. Can be used. Using a unique identification database, valid individuals can be identified from extremely few tissue specimens, whether life or death.

3. Use of GAVE6 Subsequences in Forensic Biology DNA-based personal identification techniques can also be used in forensic biology. Forensic biology is a field of science that uses genetic typing of biological evidence found in crime scenes, for example, as a means of positive identification of offenders of crime. To perform such identification, PCR technology uses DNA sequences obtained from very small amounts of biological samples found in crime scenes, such as tissues such as hair or skin, or body fluids such as blood, saliva, or semen. Is used to amplify. The amplified sequence is then compared to a standard to allow identification of the source of the biological sample.

The sequences of the present invention provide polynucleotide reagents, eg, PCR primers, that can be used to target specific loci in the human genome and increase the reliability of DNA-based forensic identification Can do. For example, the nucleic acid of interest may contain other “identification markers” (
That is, other DNA sequences that are unique to a particular individual) are provided. As described above, the actual base sequence information is used for identification as an accurate substitute for the pattern formed by the restriction enzyme fragment. The sequence targeting the non-coding region of SEQ ID NO: 1 is particularly suitable for use as the higher number of genetic polymorphisms that occur in the non-coding region, and facilitates the identification of individuals using that approach. Examples of polynucleotide reagents include GAVE6 sequences or fragments thereof, ie fragments having a length of at least 20 or 30 bases from the non-coding region of SEQ ID NO: 1.

  The GAVE6 sequence described herein is a polynucleotide reagent, ie, a labeled or labelable probe, that identifies a particular tissue, eg, brain tissue, for example, in an in situ hybridization technique Can be provided. Such polynucleotide reagents are very useful when forensic pathologists are presented with tissue of unknown origin. Such a panel of GAVE6 probes can be used to identify tissues by animal species and / or by organ type.

  In a similar manner, reagents such as GAVE6 primers or probes can be used to screen for tissue culture contaminants (ie, to screen for the presence of a mixture of atypical cells during culture).

B. Predictive Medicine
The present invention also relates to the field of predictive medicine, where diagnostic assays, prognostic assays, pharmacogenomics and clinical trial monitoring are for prognostic (predictive) purposes for the prophylactic treatment of individuals. It is used. Accordingly, one aspect of the present invention is to measure GAVE6 protein and / or nucleic acid expression and GAVE6 activity in relation to biological samples (eg, blood, urine, feces, sputum, serum, cells and tissues). It relates to a diagnostic assay. This assay can be used to determine whether an individual has a disease or disorder associated with abnormal GAVE6 expression or activity, or whether the disorder is at risk.

  The present invention also provides a prognostic (or predictive) assay for determining the risk of spreading a disorder associated with GAVE6 protein, nucleic acid expression, or activity in an individual. For example, a mutation in the GAVE6 gene can be measured in a biological sample. Such assays may be used for prophylactic or predictive purposes to treat individuals prophylactically prior to the occurrence of a disorder associated with or characterized by GAVE6 protein, nucleic acid expression, or activity. it can.

  Another aspect of the present invention is the measurement of GAVE6 protein, nucleic acid expression, or activity in an individual, whereby an appropriate therapeutic or prophylactic agent can be selected for that individual (where “pharmacogenomics” Provide a method.

  Agents for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (eg, the individual's genotype examined to determine the ability of the individual to respond to a particular drug) by pharmacogenomics (For example, a pharmaceutical product) can be selected.

  Furthermore, another aspect of the invention relates to monitoring the effects of agents (eg, pharmaceuticals or other compounds) on GAVE6 expression or activity in clinical trials. These and other agents are described in more detail in the following sections.

1. Diagnostic Assays An exemplary method for detecting the presence or absence of GAVE6 in a biological sample is to obtain a biological sample from a subject, the biological sample and the GAVE6 protein, or a nucleic acid encoding a GAVE6 protein (eg, mRNA or Genomic DNA) is contacted with a detectable compound or reagent and the presence of GAVE6 in the biological sample is detected. A preferred reagent for detecting GAVE6 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing with GAVE6 mRNA or genomic DNA. The nucleic acid probe is a full length GAVE6 nucleic acid such as, for example, the nucleic acid of SEQ ID NO: 1, or has a portion thereof having a nucleotide length of, for example, at least 15, 30, 50, 100, 250 or 500 or more. Oligonucleotides that are sufficient to specifically hybridize with GAVE6 mRNA or genomic DNA under stringent conditions. Other suitable probes for use in the diagnostic assays of the present invention are described herein.

A special reagent for detecting GAVE6 is an antibody capable of binding to the GAVE6 protein, preferably an antibody having a detectable label. The antibody is polyclonal, chimeric, and more preferably monoclonal. An intact antibody or fragment thereof (eg, _Fab or F _{(ab ') 2} ) is used. The term “biological sample” is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids within a subject's body. That is, the detection method of the present invention is used to detect GAVE6 mRNA, protein or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro detection methods for GAVE6 mRNA include Northern hybridization and in situ hybridization. In vitro detection methods for GAVE6 protein include ELISA, Western blotting, immunoprecipitation, and immunofluorescence. Methods for detecting GAVE6 genomic DNA in vitro include Southern hybridization. Further, in vivo detection methods for GAVE6 protein include introduction of labeled anti-GAVE6 antibody into a subject. For example, the antibody can be labeled with a radioactive marker and its presence and location in a subject can be detected with standard imaging techniques.

  In one embodiment, the biological sample contains protein molecules from the subject. Alternatively, the biological sample can contain mRNA molecules from the subject or genomic DNA molecules from the subject. In particular, the biological sample used here is a peripheral blood leukocyte sample separated from a subject by a conventional method.

  Therefore, carrier and patient diagnosis due to the association of disease with genetic polymorphisms of nucleic acids or proteins is advantageous for developing prognostic or diagnostic assays. For example, it would be beneficial to have a prognostic or diagnostic assay for rheumatoid arthritis, asthma, Crohn's disease, etc. GAVE6 expression is elevated intracellularly in association with activation or inflammatory conditions. Diseases related to inflammation include anaphylactic conditions, colitis, Crohn's disease, edematous conditions, contact hypersensitivity, allergies, other types of arthritis, meningitis, and other immune systems such as vasodilation, fever As well as pathological conditions that respond to damage caused by swelling and the like by locally concentrating cells, fluids, and the like. Therefore, disorders of GAVE6 metabolism can be used for diagnosis of rheumatoid arthritis. Furthermore, the molecular mechanism of rheumatoid arthritis can be detected, for example, diagnostic SNP, RFLP, variable expression level, variable function, etc., and can be detected in a tissue sample such as a blood sample.

  In other embodiments, the method further obtains a biological sample from a control subject, and contacts the control sample with a reagent that can detect the compound or GAVE6 protein, mRNA or genomic DNA, and the GAVE6 protein, mRNA or genomic DNA in the biological sample. Is then detected and then the presence and amount of GAVE6 protein, mRNA or genomic DNA in the control sample is compared to the presence and amount of GAVE6 protein, mRNA or genomic DNA in the test sample.

Chemical Library High Throughput Assay Any assay for compounds that can modulate GAVE6 activity is applicable to high throughput screening. A commercially available high-throughput screening system can be used. (See, for example, Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA). These systems typically involve the entire process of pipetting all samples and reagents, liquid dispensing, time-controlled reactions, and finally reading the microplate in a suitable detector for the assay. Has been automated. These configurable systems offer high flexibility and customization while being highly efficient and quick to launch. Such manufacturers offer various high-throughput detailed protocols. Thus, for example, Zymark Corp. provides a technical bulletin describing screening systems that detect regulation of gene transcription, ligand binding, and the like.

Kit The present invention also includes a kit for detecting the presence of GAVE6 in a biological sample (test sample). Such kits can be used to determine whether a subject has a disease associated with abnormal expression of GAVE6 (eg, an immunological disease) or is at increased risk of developing it. . For example, the kit includes a labeled compound or reagent capable of detecting GAVE6 protein or mRNA in a biological sample, and a means for measuring the amount of GAVE6 in the sample (eg, an anti-GAVE6 antibody or oligonucleotide probe comprising GAVE6 Encoding DNA (for example, that can bind to SEQ ID NO: 1) can be included. The kit also determines whether the subject is suffering from a disease associated with abnormal expression of GAVE6 or is at increased risk of developing, whether the amount of GAVE6 protein or mRNA is above or below normal levels. Can be used to get results for judgment.

  For antibody-based kits, the kit can be, for example: (1) a primary antibody that binds to the GAVE6 protein (eg, bound to a solid phase); and (2) secondary alternatives in some cases Antibodies that bind to the GAVE6 protein or primary antibody and are conjugated to a detectable reagent. If no secondary antibody is present, the primary antibody can be detectably labeled, or alternatively, molecules that bind to other primary antibodies can be detectably labeled. In any case, the label binding moiety includes one that functions as a detectable reporter molecule as is known in the art.

  With respect to oligonucleotide-based kits, the kit of the invention includes, for example, (1) an oligonucleotide, eg, a detectably labeled oligonucleotide that hybridizes to a GAVE6 nucleic acid sequence, or (2) a set of primers useful for amplifying the GAVE6 nucleic acid molecule.

  The kit can also include, for example, a buffer, a preservative, or a protein stabilizer. The kit can also include components necessary for detecting a detectable reagent (eg, an enzyme or a substrate). In addition, the kit can also include a single control sample or a set of control samples that can be measured relative to the test sample. The components of each kit are usually enclosed in separate containers, and all containers are in one package. Instructions may also be included to determine if the subject has a disease associated with abnormal expression of GAVE6 or whether it is at increased risk of developing.

2. Prognostic Assay
The methods described herein can further be used for diagnostic purposes to identify patients who have or are at increased risk of developing a disease or disorder associated with abnormal expression or activity of GAVE6. It can be used as a predictive assay. Assays described herein, such as pre-diagnostic or prognostic assays, identify patients who have or are at increased risk for developing GAVE6 protein, nucleic acid expression or activity Available for. For example, recent contact with bacterial infections or inflammation associated with asthma, chronic obstructive pulmonary disease and rheumatoid arthritis are of interest. Instead, prognostic assays can be utilized to identify patients who have a disease or disorder or are at increased risk of developing it.

  Accordingly, the present invention provides a method in which a test sample is obtained from a subject and GAVE6 protein or nucleic acid (eg, mRNA or genomic DNA) is detected. The presence of GAVE6 protein or nucleic acid diagnoses a patient who has or is at increased risk of developing a disease or disorder associated with abnormal GAVE6 expression or activity. As used herein, “test sample” means a biological sample obtained from a subject of interest. For example, the test sample is a biological fluid (eg, serum), a cell sample or tissue.

  Furthermore, the prognostic assays described herein can be used to treat drugs (eg, agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids) to patients to treat diseases or disorders associated with abnormal GAVE6 expression or activity. , Small molecules, or other drug candidate compounds) can be used. For example, such methods can be used to determine whether a patient can be effectively treated with a particular drug or class of drugs (eg, a drug that reduces GAVE6 activity). Thus, the present invention can be used to determine whether a patient can be effectively treated with an agent for a disease associated with abnormal GAVE6 expression or activity, in which a test sample is obtained and the GAVE6 protein Or nucleic acid is detected (eg, where the presence of GAVE6 protein or nucleic acid is a diagnosis that the patient can administer the agent to treat a disease associated with abnormal GAVE6 expression or activity).

  The methods of the invention can also be used to detect genetic damage or mutations in the GAVE6 gene, whereby subjects with such impaired genes are characterized by abnormal cell proliferation and / or differentiation. It can be determined whether or not there is a risk of a disease. In a preferred embodiment, the method comprises genetic damage characterized by at least one change that affects the integrity of the gene encoding the GAVE6 protein or misexpression of the GAVE6 gene in a cell sample from the subject. Including detecting the presence or absence of mutations. For example, such genetic damage or mutation can be detected by confirming the presence of at least one of the following: 1) deletion of one or more nucleotides from the GAVE6 gene; 2) addition of one or more nucleotides to the GAVE6 gene 3) substitution of one or more nucleotides in the GAVE6 gene; 4) rearrangement of the chromosome containing the GAVE6 gene; 5) variation in the level of the messenger RNA transcript of the GAVE6 gene; 6) abnormal modification of the GAVE6 gene, eg 7) unnatural level of GAVE6 protein; 8) loss of allele of GAVE6 gene; and 9) inappropriate post-translational modification of GAVE6 protein. As described herein, many assay techniques known in the art can be used to detect damage to the GAVE6 gene. A preferred biological sample is a peripheral blood leukocyte sample separated from a subject by conventional methods.

In certain embodiments, damage detection involves the use of probes / primers, which are polymerase chain reactions (PCR) such as anchor PCR or RACE PCR (see, eg, US 4,683,195 and 4,683,202), or alternatively Ligation Chain Reaction (LCR) (eg, 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 for detecting point mutations in the GAVE6 gene (eg Abravaya et al., Nucleic Acids Res (1995) 23 Volume: pages 675-682). The method includes the following steps: collection of a cell sample from a patient, separation of a nucleic acid (eg, genome, mRNA, or both) from a sample cell, a nucleic acid sample and one or more primers (high for GAVE6 gene). Contact with a primer that specifically hybridizes under conditions that allow hybridization and amplification of the GAVE6 gene (if present), and then detect the presence or absence of the amplified product or detect the size of the amplified product Compare with the length of the control sample. It is expected that PCR and / or LCR will be desirably used in the pre-amplification step along with the methods used to detect the mutations described herein.

  Alternative amplification methods include the following: self-sustained sequence replication (Guatelli et al., Proc Natl Acad Sci USA (1990) 87: 1874-1878), transcription amplification system method (Kwoh et al., Proc Natl Acad Sci USA (1989) 86: 1173-1177), Q-β replicase method (Lizardi et al., Bio / Technology (1988) 6: 1197), or all other nucleic acid amplification methods Followed by detection of molecules amplified by methods well known to those skilled in the art. This detection scheme is particularly effective when detecting nucleic acid molecules, where only a very small number of such molecules are present.

  In an alternative embodiment, mutations in the GAVE6 gene from the sample cells can be identified by changes in the restriction enzyme cleavage pattern. For example, sample and control DNA are separated, amplified (if necessary), cleaved with one or more restriction enzymes, and fragment length sizes are measured and compared by gel electrophoresis. The difference in fragment length size between the sample and control DNA indicates a mutation in the sample DNA. In addition, the use of sequence specific ribozymes (see, eg, US Pat. No. 5,498,531) can also be used to detect the presence of specific mutations by the generation or disappearance of ribozyme cleavage sites.

  In other embodiments, GAVE6 gene mutations can be identified by hybridizing with sample and control nucleic acids (eg, DNA or RNA) and high density arrays containing hundreds to thousands of oligonucleotide probes. (Cronin et al., Human Mutation (1996) 7: 244-255; Kozal et al., Nature Medicine (1996) 2: 753-759). For example, genetic mutations in GAVE6 can be identified by a two-dimensional array (as described in Cronin et al., Supra) that includes light-generated DNA probes. Briefly, the initial hybridization array of probes uses a long stretch of sample and control DNA to identify base mutations between sequences by generating a linear array of probes with overlapping sequences. Can be used when scanning. This step allows the identification of point mutations. A step followed by a second hybridization array allows the characterization of specific mutations using a smaller, more specific probe array that is complementary to all variants or detected mutations To. Each mutation array is composed of parallel probe sets (one complementary to the native gene and the other complementary to the mutant gene).

  In yet another embodiment, any of a variety of sequence analysis reactions known in the art can be used to directly read the sequence of the GAVE6 gene and to match the native (control) sequence corresponding to that of the sample GAVE6; The mutation can be detected by comparing. Examples of sequence analysis reactions include techniques developed by Maxam & Gilbert (Proc Natl Acad Sci USA (1977) 74: 560) or Sanger (Proc Natl Acad Sci USA (1977) 74: 5463). Including those based on. Various automated sequence analysis methods can also be used when performing a diagnostic assay (Bio / Techniques (1995) 19: 448), including sequence analysis by mass spectrometry (eg, 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 for detecting mutations in the GAVE6 gene include methods that can be used to detect mismatched bases in RNA / RNA or RNA / DNA heteroduplexes by protection from cleavage reagents (Myers et al., Science (1985). 230): 1242). In general, the technique of “mismatch cleavage” is to hybridize (labeled) RNA or DNA containing a native GAVE6 sequence with RNA or DNA that may have a mutation obtained from a tissue sample. There is a need to provide the heteroduplexes that are formed. This duplex is treated with a reagent that cleaves the single-stranded region of the duplex (eg, which may be present due to a base pair mismatch between the control and sample strands).

  RNA / DNA duplexes can be treated with RNase to cleave their mismatch regions, and DNA / DNA hybrids can be treated with S1 nuclease to cleave their mismatch regions. In other embodiments, either DNA / DNA or RNA / DNA duplexes can be treated with hydroxylamine or osmium tetroxide and then treated with piperidine to cleave the mismatch region. After cleavage of the mismatch region, the resulting product can be separated on a denaturing polyacrylamide gel based on size and the position of the mutation can be determined. See, for example, Cotton et al., Proc Natl Acad Sci USA (1988) 85: 4397; Saleeba et al., Methods Enzymol (1992) 217: 286-295. In preferred embodiments, the control DNA or RNA can be labeled for detection.

  In yet another embodiment, the mismatch cleavage reaction comprises one or more proteins recognizing double-stranded DNA mismatch bases (so-called “DNA mismatch repair” enzymes), point mutations in GAVE6 cDNA obtained from cell samples. A system can be defined and used to detect and map For example, E. coli-derived mutY enzyme cleaves G / A mismatch A, and Hera cell-derived thymidine DNA glycosylase cleaves G / T mismatch T (Hsu et al., Carcinogenesis (1994) 15: 1657-1662 page). In accordance with an exemplary embodiment, a probe based on a GAVE6 sequence (eg, a native GAVE6 sequence) is hybridized with cDNA or other DNA product from a test cell. The duplex is treated with a DNA mismatch repair enzyme and the cleavage product, if any, can be detected by electrophoresis procedures or the like (see, eg, US Pat. No. 5,459,039).

  In other embodiments, electrophoretic mobility changes could be used to identify mutations in the GAVE6 gene. For example, single-strand conformation polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and natural nucleic acids (Orita et al., Proc Natl Acad Sci USA). (1989) 86: 2766; Cotton, Mutat Res (1993) 285: 125-144; Hayashi, Genet Anal Tech Appl (1992) 9: 73-79. ). Single stranded DNA fragments of the sample and control GAVE6 nucleic acids are denaturated and then renatured. The secondary structure of single-stranded nucleic acids varies with sequence, and the resulting change in electrophoretic mobility can be detected even with a single base change. This DNA fragment can be labeled or detected with a labeled probe. The sensitivity of the assay is increased by using RNA (rather than DNA) because the secondary structure of RNA is more sensitive to sequence changes. In a preferred embodiment, the method utilizes a heteroduplex analysis method that splits double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al., Trends Genet (1991). 7: 5).

  In yet another embodiment, the mobility of mutant or natural fragments in a polyacrylamide gel containing a gradient of denaturant can be measured using denaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature (1985). Year) 313: 495). When DGGE is used as an analytical method, it can be modified to ensure that the DNA is not completely denatured, for example, by adding about 40 bp high melting point GC-rich DNAGC clamp by PCR. In a further embodiment, a temperature gradient can be used instead of a denaturant gradient to identify differences in mobility of sample and control DNA (Rosenbaum et al., Biophys Chem (1987) 265: 12753).

  Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers can be made to hybridize to a target DNA under conditions that allow hybridization only if a known mutation is located in the middle and an exact match is found (Saiki et al. Nature (1986) 324: 163); Saiki et al., Proc Natl Acad Sci USA (1989) 86: 6230). Such allele-specific oligonucleotides hybridize to a number of different variants when bound to PCR-amplified target DNA and when the oligonucleotide is bound to a hybridizing membrane and hybridized to labeled target DNA. .

  Alternatively, allele specific amplification technology which depends on selective PCR amplification can also be used with the present invention. Oligonucleotides used as primers for specific amplification can carry mutations of interest in the middle part of the molecule (for amplification depending on differential hybridization) (Gibbs et al., Nucleic Acids Res (1989) 17: 2437-2448), or with primers at the extreme 3 'end, mismatches can prevent or reduce the polymerase extension reaction under appropriate conditions (Prossner, Tibtech (1993) 11: 238). Furthermore, it is also desirable to create a detection method based on cleavage by introducing a new restriction enzyme recognition site in the mutation region (Gasparini et al., Mol Cell Probes (1992) 6: 1). In one embodiment, amplification can also be achieved using Taq ligase for amplification (Barany, Proc Natl Acad Sci USA (1991) 88: 189). In such cases, binding is only possible when there is a perfect match at the 3 'end of the 5' sequence, which allows detection of the presence of a known mutation at a particular site by looking for the presence or absence of amplification. Become.

  The methods described herein can also be performed, for example, with a prepackaged diagnostic kit containing at least one probe nucleic acid or antibody reagent described herein. The methods and kits can be conveniently used in the diagnosis of patients presenting with a symptom or family history of a disease or condition associated with the GAVE6 gene, for example, in a clinical setting.

  Further, any cell type or tissue in which GAVE6 is expressed can be utilized in the prognostic assay described herein.

3. Pharmacogenomics Agents or modulators that have a stimulatory or inhibitory effect on GAVE6 activity (eg, expression of the GAVE6 gene) identified in the screening assays described herein are those diseases that are associated with GAVE6 activity ( For example, it can be administered to an individual to treat (prophylactically or therapeutically) inflammation associated with asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis). Along with such treatment, the pharmacogenomics of an individual (ie, a study of the association between an individual's genotype and the individual's susceptibility to foreign compounds or pharmaceuticals) can be considered. Differences in the metabolism of a therapeutic agent can result in severe toxicity or treatment failure by altering the relationship between the blood concentration and dosage of a pharmacologically active drug. Thus, an individual's pharmacogenomics allows for the selection of effective drugs (eg, pharmaceuticals) for prophylactic or therapeutic treatments based on consideration of the individual's genotype. Such pharmacogenomics are further useful for determining appropriate dosages and treatment regimens. Thus, the activity of an individual's GAVE6 protein, the expression of a GAVE6 nucleic acid, or the content of a mutation in the GAVE6 gene can be determined by them to select an appropriate drug for the therapeutic or prophylactic treatment of the individual. .

  Pharmacogenomics deals with genetically responsive hereditary variants due to altered drug processing and abnormal effects of a diseased person of clinical significance. See, for example, Linder, Clin Chem (1997) 43 (2): 254-266. In general, two types of pharmacogenomic conditions are distinguished. A genetic condition that is inherited as a single factor that changes the mode of action of a drug on a living body is called "altered drug action". The genetic condition inherited as a single factor that changes the manner in which the body acts on pharmaceuticals is called "altered drug metabolism". Pharmacogenomic conditions occur as either rare disorders or genetic polymorphisms. For example, glucose-6-phosphate dehydrase deficiency (G6PD) is a well-known genetic enzyme disease in which the main clinical complications are oxidant drugs (antimalarial drugs, sulfa drugs, analgesics) , Or nitrofuran) after ingestion and after consumption of broad beans.

  As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug-metabolizing enzymes (eg, N-acetyltransferase 2 (NAT2) and the cytochrome P450 enzymes, CYP2D6 and CYP2C19) It has provided explanations for excessive drug reactions and why the standard shows severe toxicity after taking safe amounts of drugs. Genetic polymorphisms are expressed in populations as two phenotypes: an exogenous person (extensive metabolizer (EM)) and a slow metabolite (poor metabolizer (PM)). The incidence of PM is different in different population groups. For example, the gene encoding CYP2D6 is very polymorphic and many mutations have been identified in PM, all resulting in the absence of functional CYP2D6. Persons with slow metabolism of CYP2D6 and CYP2C19 experience enhanced drug response and side effects quite often even when receiving standard doses. If the metabolite is an active therapeutic moiety, as demonstrated by codeine's analgesic action by morphine, a metabolite produced by CYP2D6, PM does not respond to treatment. In other extreme cases, so-called ultra-rapid metabolizers, which do not respond to standard doses. Recently, it has been found that the molecular basis of very fast metabolizing is due to gene amplification of CYP2D6.

  Thus, the amount of GAVE6 protein activity, GAVE6 nucleic acid expression or GAVE6 gene mutation in an individual is measured to thereby select an appropriate pharmaceutical for the individual's therapeutic or prophylactic therapy. can do. In addition, pharmacogenomic studies can be used to apply the allelic genotypes that result in genetic polymorphisms encoding drug-metabolizing enzymes to identify an individual's drug-responsive phenotype. When this knowledge is applied to dosage and drug selection, side effects and treatment failures can be avoided and identified by GAVE6 modulators, such as one of the sample screening assays described herein. The therapeutic or preventive effect when treating a patient using the regulated substance can be enhanced.

4). Monitoring effects during clinical trials Monitoring the effects of drugs (eg, drugs or compounds) on the expression or activity of GAVE6 (eg, the ability to modulate abnormal cell growth and / or differentiation) is not possible with basic drug screening alone. It can also be applied in clinical trials. For example, the effect of a drug on GAVE6 gene expression, protein amount or protein activity, as measured by a screening assay as described herein, may be used to reduce GAVE6 gene expression, protein amount or protein activity. It can be monitored in the clinical trials of the patients shown. Instead, the effect of an agent that decreases GAVE6 gene expression, protein amount or protein activity, as measured by a screening assay, in patients exhibiting an increase in GAVE6 gene expression, protein amount or protein activity. Can be monitored during clinical trials. In such clinical trials, the expression or activity of GAVE6, and preferably the expression or activity of other genes involved in, for example, cell proliferation disorders, are used as markers of immune responsiveness of specific cells Can do. For example, but not limited to being modulated in a cell that has been treated with an agent (eg, a compound, pharmaceutical, or small molecule) that modulates GAVE6 activity (eg, identified in a screening assay described herein) Genes can also be identified, including GAVE6. Thus, for example, in clinical trials, to study the effects of drugs on cell proliferation disorders, cells are isolated, RNA is prepared, and expression levels of genes involved in GAVE6 and other disorders are analyzed. it can. Gene expression (ie, gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively, by the method described herein. It can be quantified by measuring the amount of protein produced by one or by measuring the level of activity of GAVE6 or other genes. In this way, the gene expression pattern can be used as a marker showing physiological responsiveness of a cell to a drug. Thus, the status of such a response can be measured in advance and at various times during treatment with an individual's medication.

  In certain embodiments, the invention is based on an agent (eg, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assay described herein). A method is provided for monitoring the effects of treatment on a patient, comprising the following steps: (i) collecting a pre-dose sample from the patient prior to treatment with the drug; (ii) GAVE6 in the pre-dose sample Detecting protein expression, mRNA or genomic DNA levels; (iii) collecting one or more post-administration samples from a patient; (iv) expression or activity of GAVE6 protein in the post-administration sample, mRNA Or detecting the level of genomic DNA; (v) expression or activity of GAVE6 protein, mRNA or genomic in the sample prior to administration. Comparing the level of DNA to the level of GAVE6 protein, mRNA or genomic DNA in one or more post-administration samples; and (vi) altering administration of the drug to the patient accordingly. Is included. For example, increasing the administration of a drug is desirable to increase GAVE6 expression or activity to a higher concentration than that detected, ie, to increase the effect of the drug. Instead, it is desirable to reduce the administration of the drug in order to reduce the expression or activity of GAVE6 to a lower concentration than that detected, i.e. to reduce the effect of the drug.

D. Therapeutic Methods The present invention provides both prophylactic and therapeutic methods for treating patients with or at risk for (or susceptible to) diseases associated with abnormal GAVE6 expression or activity. Such diseases include, but are not limited to, inflammatory disorders such as asthma, chronic obstructive pulmonary disease and rheumatoid arthritis.

1. Prophylactic Methods In one aspect, the present invention protects patients from diseases or conditions associated with abnormal GAVE6 expression or activity by administering to the patient an agent that modulates GAVE6 expression or at least one GAVE6 activity. Provide a method. Patients at risk of disease caused by or aberrant GAVE6 expression or activity can be identified, for example, by any of the diagnostic or prognostic assays described herein, or combinations thereof. Administration of a prophylactic agent can be performed before the symptoms characterized by abnormal GAVE6 are exacerbated, preventing the disease or disorder, or alternatively, delaying the progression. Depending on the type of GAVE6 abnormality, for example, a GAVE6 agonist or GAVE6 antagonist agent can be used to treat the patient. Appropriate agents can be determined based on screening assays described herein.

2. Therapeutic Methods Another aspect of the present invention relates to methods of modulating GAVE6 expression or activity for therapeutic purposes. The modulating method of the invention comprises contacting a cell with an agent that modulates one or more activities of GAVE6 protein activity associated with the cell. An agent that modulates GAVE6 protein activity can be an agent as described herein, eg, a nucleic acid, protein, a naturally occurring cognate ligand of the GAVE6 protein, a peptide, a GAVE6 peptidomimetic, or other small molecule. . In one example, the agent promotes one or more of the biological activities of the GAVE6 protein. Examples of such enhancers include active GAVE6 protein and nucleic acid molecules encoding GAVE6 introduced into cells. In other embodiments, the agent inhibits one or more of the biological activities of the GAVE6 protein. Examples of such inhibitors include antisense GAVE6 nucleic acid molecules and anti-GAVE6 antibodies. This method of modulation is performed in vitro (eg, by culturing cells with the drug) or alternatively in vivo (eg, by administering the drug to a patient). Accordingly, the present invention provides a method of treating an individual suffering from a disease or disorder characterized by abnormal expression or activity of a GAVE6 protein or nucleic acid molecule. In one embodiment, the method comprises an agent (eg, an agent identified by the screening assay described herein) or an agent that modulates GAVE6 expression or activity (eg, upregulate or downregulate). Administration of the combination. In other embodiments, the method also includes administering a GAVE6 protein or nucleic acid molecule as a treatment to complement reduced or abnormal GAVE6 expression or activity.

  Promotion of GAVE6 activity is desirable when GAVE6 is abnormally down-regulated and / or where increasing GAVE6 activity may have beneficial effects. Conversely, inhibition of GAVE6 activity is desirable when GAVE6 is abnormally upregulated and / or where reducing GAVE6 activity may have an advantageous effect.

  The present invention may be better understood with reference to the following, non-limiting examples provided for illustration of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. However, it should not be construed as limiting the broad scope of the invention.

Example
Methods and Materials Identification of GAVE6 A homology search against the human genome sequence database HTG (NCBI / NIH) was performed by the FASTA algorithm (Wisconsin GCG Package Version 10.1) using various GPCRs as queries. Genomic DNA sequences found to have statistical significance were translated into three forward frames for BLASTp searches of protein databases. Genomic DNA sequence AC013396 was identified as containing a sequence assumed to be a GPCR and was named GAVE6. The position of GAVE6 on the chromosome was mapped to 2p22.1.

Cloning of genomic DNA encoding GAVE6 Primers specific to the putative GAVE6 5 ′ and 3 ′ sequences were designed. The forward primer, HP157, CAG CCC ATG GAA CTT CAT AAC CTG (SEQ ID NO: 5), and the reverse primer, HP158, CTG GCC CTC AGC CCT GGG AGG AG (SEQ ID NO: 6) were used for polymerase chaining using human genomic DNA as a template. Used for amplification of GAVE6 genomic DNA by reaction (PCR). The conditions of PCR are as follows: denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension reaction at 72 ° C for 1 minute, 35 cycles, followed by extension at 72 ° C for 5 minutes reaction. The amplified DNA fragment was cloned into the pCRII-TOPO vector (obtained from Invitrogen). The cloned DNA insert was confirmed by DNA sequencing. All PCR amplifications were performed in DNA Engine Tetrad (MJ Research, model PTC-225).

Northern Blot Analysis Northern blots from multiple human tissues (obtained from Clontech) were hybridized with DNA fragments of full length open reading frames labeled with [α- ³² P] dCTP according to manufacturer's instructions. Hybridized blots were washed with 2 × SSPE and 0.1% SDS for 30 minutes at 50 ° C., followed by 0.1 × SSPE and 0.1% SDS for 1 hour at 50 ° C. The blot was then exposed to X-ray film at -70 ° C in the presence of an intensifying screen. The results of this Northern blot analysis are shown in FIG.

Taqman analysis Total RNA of human tissue was purchased from Clontech. Prior to preparation of cDNA, total RNA was subjected to DNAse1 treatment to remove possible genomic DNA contamination. Briefly, total RNA is 5 μl of 10 × DNAse1 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 of DNAse1 (RNase free) (2 U / μL; Ambion) was applied to a final volume of 50 μL and reacted at 37 ° C. for 1 hour. Following the phenol precipitation step, cDNA synthesis was performed using the Superscript choice system (as described by Life Technologies). Taqman primers / probes were designed using Primer Express 1.0 software (ABI). Taqman forward primer for GAVE6: 5 ′ GCT GCC TGC AAA GTC AAC CT 3 ′ (SEQ ID NO: 7), reverse primer: 5 ′ TGG CTG TGA GGA AGA CAA CG 3 ′ (SEQ ID NO: 8) and Taqman probe sequence: 5 ′ FAM-CCA CCA ACC GCA CGG CAA-TAMRA 3 ′ (SEQ ID NO: 9). Fam was used as a reporter dye and Tamra was used as a quencher. Taqman probes were ordered from Operon Technologies and synthesized. Taqman reactions were performed in 96-well plate MicroAmp optical tubes (optical tubes (PE)) with a final volume of 50 μL (Taqman PCR Mixture (Perkin Elmer) 25 μL; final concentration 900 nM forward primer 1 μL; final concentration 900 nM reverse primer 1 μL; And 1 μL Taqman probe at a final concentration of 200 nM; containing 5 μL cDNA template (10 ng / μL at the calculated concentration) and 17 μL water. Taqman PCR conditions were set as described in PE Applied Biosystem. Human beta actin primer probe (designed and purchased by PE applied Biosystem) was used as an internal control. For each tissue, Taqman reactions were performed twice for both the target gene and the internal control. In addition, a standard curve was generated by increasing the amount of template twice for human beta actin in whole brain cDNA. Thereby, the relative number of amplified amplification units (amplicon) can be calculated. The expression of the target gene is expressed as a relative expression multiple relative to the brain cDNA. The data obtained by Taqman analysis is shown in Table 1 and illustratively in FIG.

PCR screening of cDNA library PCR primers specific for the GAVE6 coding region: 5 'TTC CTC CTG ATC AGC AAC CT 3' (SEQ ID NO: 10), and 5 'TTG GTG GAC AGC ATG AAG AG 3' (SEQ ID NO: 11) Were used for screening pooled human spleen, brain, kidney, activated T cells, and lung cDNA libraries. PCR screening was performed in 96-well plates with the following PCR protocol: hold at 94 ° C. for 3 minutes; 40 cycles of 94 ° C. for 30 seconds, 52 ° C. for 30 seconds, and 68 ° C. for 45 seconds. Positive subpools are subsequently diluted for subsequent rounds of PCR screening. A limited number of colonies obtained from the positive subpool were plated on agar plates and positive plasmids were confirmed by PCR and then subjected to DNA sequence analysis.

  The invention is not limited to the scope of the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to persons skilled in the art from the foregoing description and accompanying data. Such modifications are also intended to fall within the scope of the appended claims.

  Furthermore, all base sizes or amino acid sizes of nucleic acids or polypeptides, and all molecular weights or molecular weight data are approximate and are provided for the specification.

  Various publications are cited herein, the entire description of which is incorporated by reference.

DNA sequence encoding GAVE6 (SEQ ID NO: 1) Amino acid sequence of GAVE6 (SEQ ID NO: 2) Comparison of the amino acid sequence of GAVE6 (SEQ ID NO: 2) with the amino acid sequences of HM74 and GPR31 (SEQ ID NO: 3 and SEQ ID NO: 4, respectively). Northern blot of transcripts from various organs of GAVE6. Expression profiles of various organs in the human organ / tissue panel of GAVE6.

Claims (29)

  An isolated nucleic acid molecule containing the DNA sequence of Figure 1 (SEQ ID NO: 1).   2. An isolated nucleic acid molecule capable of hybridizing to the isolated nucleic acid molecule of claim 1 under stringent hybridization conditions, or a complementary hybridization to the isolated nucleic acid molecule of claim 1. For the probe.   The isolated nucleic acid molecule according to claim 1 or 2, which encodes a polypeptide having the amino acid sequence of Figure 2 (SEQ ID NO: 2).   3. The isolated nucleic acid molecule of claim 2, which encodes a polypeptide having an amino acid sequence at least 30% equal to the amino acid sequence of SEQ ID NO: 2.   3. An isolated nucleic acid molecule according to claim 1 or 2 that is detectably labeled.   6. The isolated nucleic acid molecule of claim 5, wherein the detectable label contains an enzyme, a radioisotope, or a fluorescent chemical.   A purified polypeptide containing the amino acid sequence of FIG. 2 (SEQ ID NO: 2).   8. An isolated nucleic acid molecule encoding the purified polypeptide of claim 7. 8. The purified polypeptide of claim 7, which is detectably labeled.   The purified polypeptide of claim 9, wherein the detectable label contains an enzyme, a radioisotope, or a fluorescent chemical.   An antibody having the purified polypeptide according to claim 7 as an immunogen.   12. The antibody of claim 11, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, or a chimeric antibody.   12. The antibody of claim 11, wherein the antibody is detectably labeled.   14. A purified polypeptide according to claim 13, wherein the detectable label contains an enzyme, a radioisotope, or a fluorescent chemical.   2. An expression vector containing the isolated nucleic acid molecule of claim 1 operably linked to an expression control element.   3. An expression vector containing the isolated nucleic acid molecule of claim 2 operably linked to an expression control element.   The expression vector according to claim 15 or 16, wherein the expression control element is selected from the group consisting of a constitutive control sequence, a cell-specific control sequence, and an inducible control sequence.   The expression vector according to claim 17, wherein the expression control element is a promoter. Promoters are hCMV immediate early promoter, early promoter of SV40, early promoter of adenovirus, early promoter of vaccinia, early promoter of polyoma, late promoter of SV40, late promoter of adenovirus, late promoter of vaccinia, late promoter of polyoma , Lac system, trp system, TAC system, TRC system, lambda phage major operator and promoter, fd coat protein regulatory region, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or yeast alpha mating factor promoter The expression vector according to claim 18.   A host cell transformed or transduced with the expression vector according to claim 15 or 16.   21. The host cell of claim 20, wherein the host cell contains a prokaryotic cell or a eukaryotic cell.   The host cell according to claim 21, wherein the host cell is an E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cell. Process,
a) culturing the host cell of claim 20 under conditions allowing expression of the isolated polypeptide; and
The method for producing an isolated polypeptide according to claim 7, comprising b) recovering the isolated polypeptide from the host cell, the culture medium, or a mixture thereof.   A therapeutic method for modulating GAVE6 signaling activity or signaling, comprising administering to a patient an agonist, antagonist or inverse agonist of GAVE6 in a patient in need of treatment. The following steps:
Contacting an agonist candidate substance with a GAVE6-expressing cell; and
Measuring whether the signal activity of GAVE6 in the presence of the agonist candidate substance is increased compared to the signal activity of GAVE6 in the absence of the agonist candidate substance;
A method of identifying an agonist of GAVE6. The following steps:
Contacting an inverse agonist candidate substance with a GAVE6-expressing cell; and
Whether the signal activity of GAVE6 is decreased in the presence of the inverse agonist candidate substance compared to the signal activity of GAVE6 in the absence of the inverse agonist candidate substance, and decreased in the presence of the endogenous ligand or agonist Measuring whether or not
A method for identifying an inverse agonist of GAVE6. The following steps:
Contacting an antagonist candidate substance with a GAVE6-expressing cell; and
Determining whether the signal activity of GAVE6 in the presence of said antagonist candidate substance is reduced compared to the signal activity of GAVE6 in the presence of an endogenous ligand or agonist;
A method for identifying antagonists of GAVE6.   A therapeutic composition comprising an agonist, antagonist, or inverse agonist of GAVE6 that can modulate GAVE6 signaling activity or transmission.   A method of treating a disease, comprising administering to a patient in need of treatment a therapeutic composition containing an agonist, antagonist or inverse agonist of GAVE6 that can modulate GAVE6 signaling activity or signal transduction.

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