JP2005517398A - Nucleic acid encoding G protein-coupled receptor and use thereof - Google Patents

Abstract

Provided here are novel and useful G protein-coupled receptors that are involved in signal transduction for inflammatory and physiological immune responses. Also provided are methods of using the receptor to screen for compounds that can modulate the activity of the receptor. Such compounds are readily available for the treatment of many inflammatory and immune related diseases and disorders.

Description

  The present invention relates generally to novel nucleic acid molecules encoding GAVE19, a previously unknown G protein-coupled receptor, and the use of such nucleic acid molecules and GAVE19.

  G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that are involved in intracellular signaling. GPCRs respond to, transform, and initiate a second messenger response within the cell in response to various extracellular signals including neurotransmitters, hormones, odorants and light. Many therapeutic agents target GPCRs because these receptors mediate a wide variety of physiological responses including inflammation, vasodilation, heart rate, bronchodilation, endocrine and peristalsis. is there.

GPCRs are characterized by an extracellular domain, a 7-transmembrane domain, and an intracellular domain.
Some of the actions performed by the receptor, such as ligand binding and interaction with G proteins, are related to the presence of certain amino acids in critical positions. For example, various studies have revealed that amino acid sequence differences in GPCRs cause differences in affinity for either natural ligands or small molecule agonists or antagonists. In other words, minor differences in the sequence cause differences in binding affinity and activity (eg Meng et al., J. Biol. 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 (1): 413-21). In particular, several studies have revealed that amino acid sequence differences in the third intracellular domain result in activity differences. Myburgh et al. Found that alanine 261 in the intracellular loop 3 of the gonadotropin-releasing hormone receptor is crucial for G protein coupling and receptor internalization (receptor-dependent endocytosis). (Biochem J (1998) 331 (Part3): 893-6). Wonerow et al. Studied the thyrotropin (thyroid stimulating hormone) receptor and demonstrated that the loss of the intracellular third loop affects the structural receptor activity (J Bio Chem (1998) 273 (14): 7900 -Five).

In general, the binding of an endogenous ligand to a receptor causes a change in the conformation of the intracellular domain of the receptor, allowing conjugation between the intracellular domain and intracellular substances (eg G protein) Result. . The G _{protein, G q, G s, G} i, there are several things like G _z and G _o (e.g., Dessauer et al, Clin Sci ( Colch) (1996) 91 (5):
527-37).
The IC-3 loop interacts with the G protein as well as the carboxy terminus of the receptor (Pauwels et al., Mol Neurobiol (1998) 17 (1-3): 109-135 and Wonerow et. Al., Supra). ). Certain GPCRs have a mixed relationship with the G protein. That is, one GPCR can interact with one or more G proteins (see, for example, Kenakin, Life Science (1988) 43: 1095).

  The GPCR activated by the ligand couples with the G protein to initiate a signal cascade (referred to as “signal transduction (signal amplification)”). Such signaling ultimately results in cellular activity or inhibition.

GPCRs exist in the cell membrane in equilibrium between two different conformations: “inactive” and “active”. Inactive receptors cannot be linked to intracellular signaling pathways that cause biological responses (an exception is seen during overexpression of receptors in transmitting cells, eg www.Creighton.edu/Pharmacology/inverse .htm. )

By adjusting the conformation to the active state, it is possible to link to the transmission pathway (via the G protein) and generate a biological response. It is most likely that the active conformation results from the binding of the agonist. However, often there is already a significant degree of response in the absence of any agonist, and such receptors are called constitutive activity (i.e. in an already active conformation). Is independent of ligand, or autonomously active). When an agonist is added to such a system, an increase in response is usually observed. However, when a classical antagonist is added, binding by such molecules has no effect. On the other hand, certain antagonists result in inhibition of constitutive activity of the receptor, suggesting that the latter belongs to an agonist with negative intrinsic activity rather than an antagonist by strict interpretation. ing. These drugs are called inverse agonists ( www.Creighton.edu/Pharmacology/inverse.htm. ).

Traditional research on receptors has begun on the premise that endogenous ligands should be identified first before the discovery of performing antagonist and other receptor effector molecules (effect molecules). The doctrine response is to identify the endogenous ligand, even if the antagonist may be discovered earlier (WO 00/22131).
. However, since the active state is most useful for assay screening purposes, obtaining a receptor with constitutive activity, particularly a GPCR, is an agonist, partial agonist, inverse agonist in the absence of information about endogenous ligands. And allows easy isolation of antagonists. Furthermore, in diseases resulting from impaired receptor activity, drugs that cause inhibition of constitutive activity or more specifically reduce the concentration of effectively activated receptors are autonomous. It seems that it can be found more easily by assay using receptors in the active state. For example, in the case of receptors transfected into patients for the treatment of disease, it is contemplated that the activity of such receptors can be fine-tuned by inverse agonists discovered by the assay.

  Diseases such as asthma, chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis (RA) are generally thought to be due to inflammatory pathogenesis involving helper T cells, monocyte macrophages and eosinophils. Today's anti-inflammatory treatments with steroids are effective for asthma, but with metabolic and endocrine side effects. The same is probably true for inhalation formulations that are absorbed through the lung or nasal mucosa. There is currently no satisfactory oral treatment for RA or COPD.

Eosinophils mediate the majority of airway dysfunction in allergies and asthma. Interleukin 5 (IL-5) is a cytokine that activates and activates eosinophils.
Studies have shown that IL-5 is essential 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 T cell-2 (Th2) in atopic asthma upon exposure to allergens (eg, house dust mite antigen).

  RA is believed to be the result of accumulation of activated macrophages in the affected synovium. Interferon γ (IFNγ) is a cytokine derived from helper T cell-1 (Th1) having various pro-inflammatory properties. It is the most potent macrophage activating cytokine and induces MHC class II gene transcription that contributes to a dendritic cell-like phenotype.

Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria and elicits an inflammatory response including tumor necrosis factor alpha (TNFα) release.
The efficacy of intravenous anti-TNFα treatment in RA has been clinically demonstrated. COPD is also believed to be the result of macrophage accumulation in the lung, which produces neutrophil chemoattractants (eg, IL-8: de Boer et al., J Pathol (2000) 190 (5 ): 619-626). Both macrophages and neutrophils release cathepsins, causing alveolar wall collapse. It is believed that the lung epithelium can be an important material for 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): 2205- 2213).

  Identifying and characterizing previously unknown GPCRs can be useful for disease states involving GPCR activity if the GPCR is involved in the disease and has the potential to treat the disease by modulating the activity of the GPCR. Development of new compositions and methods for treatment can be provided. 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 previously unknown GPCRs to identify compounds that can act as potential agonists or antagonists for GPCRs. These compounds are readily applicable as therapeutic agents for modulating GPCR activity in vivo and can therefore be used in the treatment of many diseases related to GPCR activity.

  Any document cited herein does not imply that it has been adopted as possible as a “prior art document” that can be applied immediately.

  The present invention provides novel constitutively active murine GPCRs, GAVE19, and elucidates their expression characteristics and provides compositions and methods applied in the discovery for related disease identification and treatment Is.

  Thus broadly, the present invention extends to an isolated nucleic acid molecule comprising the DNA sequence of FIG. 1 (SEQ ID NO: 1), a variant thereof, a fragment thereof, or an analogue or derivative thereof. Is. Variants of the invention include allelic variants, degenerate gene variants or allelic variants as a result of degenerate changes in sequence.

Furthermore, the present invention extends to an isolated nucleic acid molecule of SEQ ID NO: 1, or an isolated nucleic acid molecule that can hybridize with a variant thereof under stringent hybridization conditions. Moreover, the present invention extends to an isolated nucleic acid molecule that can hybridize under stringent hybridization conditions with a nucleic acid molecule complementary to the DNA sequence of SEQ ID NO: 1. Stringent hybridization conditions will be described later.
The present invention further extends to an isolated nucleic acid molecule comprising a DNA sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

In some cases, the isolated nucleic acid molecules of the invention described above may be detectably labeled. Examples used herein as detectable labels include, but are not limited to, enzymes, radioisotopes, or fluorescent chemicals. Specific examples of detectable labels are described below.

  Certain polypeptides are also encompassed by the present invention. For example, the invention extends to a purified polypeptide comprising the amino acid sequence of SEQ ID NO: 2, a conservative variant thereof, or an analog or derivative thereof. In some cases, the polypeptides of the invention may be detectably labeled.

  In addition, the present invention extends to an antibody when the polypeptide of the present invention is an immunogen used for antibody production. These antibodies may be monoclonal or polyclonal. Furthermore, the antibody may be “chimeric” and may, for example, contain the protein domain of an antibody raised against a purified polypeptide according to the invention in a different species. Of course, the antibody according to the present invention may be detectably labeled. Specific examples of detectable labels used herein are described below.

  The present invention further extends to an expression vector comprising a nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1, a variant thereof, an analogue or derivative thereof, or a fragment to which an expression control factor is operatively associated. It is. Moreover, the expression vector according to the present invention is a single molecule capable of hybridizing under stringent hybridization conditions with an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1 in which an expression control factor is operatively associated. A hybridization probe that may comprise a separated nucleic acid molecule or that is complementary to an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1, wherein the hybridization probe is operable with an expression regulator Can be hybridized under stringent hybridization conditions. A special example of an expression control element used here is a promoter. Examples of special promoters that can be used in the present invention include 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 system, trp system, TAC system, TRC system, lambda phage major operator and promoter region, fd coat protein regulatory region, 3-phosphoglycerate kinase promoter, acid phosphatase A promoter or a promoter of yeast α-mating factor can be mentioned, but is not limited thereto.

  The expression vector according to the present invention can be used to transfect or transform a host cell and produce a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof. The host cell can be either prokaryotic or eukaryotic. Specific examples of single cells used here include E. coli Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1.1, BW, L -M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells etc. are included.

  Furthermore, the present invention extends to 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 include culturing a transformed or transfected host cell using the expression vector according to the present invention under conditions suitable for expression of the purified polypeptide, and treating the purified polypeptide with a unicellular host, host It consists of collecting from the cell culture medium or both.

The present invention also extends to assays for identifying compounds that can modulate the activity of GAVE19. Such a compound can be an agonist, antagonist or inverse agonist of GAVE19. Therefore, the present invention provides GAV in the presence of endogenous ligands.
Contacting a cell expressing E19 with a potential agonist, and whether the signaling activity of GAVE19 in the presence of the potential agonist is increased compared to the signaling activity of GAVE19 in the absence of the potential agonist It also extends to a method for identifying agonists of GAVE19 comprising measuring.

  Similarly, the present invention extends to a method for identifying inverse agonists of GAVE19. The method involves contacting a potential inverse agonist with a cell expressing GAVE19, and the signaling activity of GAVE19 in the presence of the potential inverse agonist and endogenous ligand or agonist is present in the presence of the endogenous ligand or agonist. It consists in determining whether it decreases compared to the signaling activity of GAVE19 under conditions that are free of potential inverse agonists.

  Of course, the present invention extends to methods for identifying antagonists of GAVE19. The method includes contacting a potential antagonist with a cell expressing GAVE19, and the signaling activity of GAVE19 in the presence of the potential antagonist is compared to the activity of GAVE19 in the presence of an endogenous ligand or agonist. It consists of the process of measuring whether it decreases or not.

Therefore, one aspect of the present invention provides an isolated nucleic acid sequence encoding a GAVE19 protein, fragment thereof or variant thereof.
One aspect of the present invention also provides a variant of a nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1 and a DNA molecule capable of hybridizing to SEQ ID NO: 1 under stringent conditions.

Furthermore, one aspect of the present invention provides an amino acid sequence of GAVE19, a variant thereof, a fragment thereof, or an analogue or derivative thereof.
Furthermore, one aspect of the present invention provides an expression vector comprising a DNA sequence encoding GAVE19, a variant thereof, a fragment thereof or an analogue or derivative thereof (the DNA sequence is operatively associated with an expression regulator). It is to provide.
Still further, the present invention provides an antibody having GAVE19, a variant thereof, an analog or derivative thereof, or a fragment thereof as an immunogen.

  Furthermore, the present invention also includes a method for identifying compounds capable of modulating the GAVE19 protein. Such modulator may be an antagonist of GAVE19, an agonist of GAVE19 or an inverse agonist of GAVE19. Moreover, compounds that modulate GAVE19 expression or activity in mice treat many diseases or disorders such as various inflammatory diseases, asthma, chronic obstructive pulmonary disease (COPD), and rheumatoid arthritis. May be used to do.

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

[Detailed explanation]
As described above, the present invention relates to the surprising and surprising discovery of a previously unknown mouse nucleic acid molecule that encodes a previously unknown G protein coupled receptor, designated herein as GAVE19. In particular, surprisingly, the fact that GAVE19 is expressed in immune tissues or organs such as kidney, liver and small intestine has become apparent. Therefore, GAVE19 is immediately useful as a target for the development of pharmaceutical compositions for the treatment of various inflammatory diseases such as asthma, rheumatoid arthritis, COPD and the like.

Various terms and phrases used throughout the detailed description and claims to describe the invention are as follows:
As used herein, the term “modulator” refers to those that modulate the activity of GAVE19 (eg, but not limited to, ligands and candidate compounds). As the modulator (regulator) of the present invention, an agonist, partial agonist, antagonist or inverse agonist of GAVE19 can be considered.

  As used herein, the term “agonist” refers to one that activates an intracellular response when bound to a receptor, or one that increases GTP binding to a membrane (eg, but not limited to a ligand and a candidate compound). ).

  The term “partial agonist” as used herein activates an intracellular response when bound to a receptor, but to a lesser extent / range compared to an agonist, or increases GTP binding to the membrane. Refers to those whose degree / range is small compared to an agonist (eg, but not limited to ligands and candidate compounds).

  The term “antagonist” as used herein refers to those that competitively bind to a receptor at the same site as the agonist binds (eg, but not limited to ligands and candidate compounds). However, antagonists do not activate the intracellular response initiated by the activated form of the receptor and can therefore inhibit the intracellular response by agonists or partial agonists. In a related aspect, the antagonist does not reduce the baseline intracellular response in the absence of an agonist or partial agonist.

  As used herein, the term “inverse agonist” refers to those that bind to structurally active receptors and inhibit baseline intracellular responses (eg, but not limited to ligands and candidate compounds). . A baseline response is observed when no agonist or partial agonist is present or when there is little GTP binding to the membrane and is initiated when the active form of the receptor is below the normal activity reference value.

  As used herein, the term “candidate compound” refers to a compound (for example, but not limited to, a chemically synthesized compound) applicable to a screening technique. In certain embodiments, the term does not include those known as compounds selected from the group consisting of agonists, partial agonists, inverse agonists or antagonists of GAVE19. These compounds have been identified by traditional drug discovery methods, which include identification of endogenous ligands specific for the receptor and / or screening of candidate compounds for the receptor, Such screening requires a competitive assay to assess the effect.

  As used herein, the terms “structurally activated receptor” or “autonomously active receptor” are used herein interchangeably and the presence of a ligand It means a receptor that is subject to activation under non-operating conditions. Such structurally activated receptors are endogenous (eg GAVE19), or non-endogenous, ie in the case of GPCRs, modified by recombinant technology to produce a structural form by mutation of the wild type GPCR. (See, eg, EP 1071701; WO 00/22129; WO 00/22131; and US Pat. Nos. 6,150,393 and 6,140,509, which are hereby incorporated by reference in their entirety).

  As used herein, the term “constitutive receptor activation” means the stabilization of the receptor in the active state by a method other than receptor binding with an endogenous ligand or its chemical equivalent. To do.

  As used herein, the term “ligand” refers to those that bind to other molecules, including but not limited to hormones or neurotransmitters, and further to receptors. Those that bind with steric selectivity.

  As used herein, the term “family” as used for the protein or nucleic acid of the invention is an amino acid or nucleotide sequence that has a structural domain that is common in appearance and that is sufficient as defined herein. Means two or more proteins or nucleic acid molecules having the same identity. Such family members are naturally occurring and are derived from both the same or different species. For example, one family includes a first protein of human origin and a homologue of the mouse origin of that protein, as well as another protein of human origin as a second protein and a homologue of that second protein of mouse origin. Can do. One family member may also have common functional characteristics.

As used herein, the terms “GAVE19 activity”, “biological activity of GAVE19”, and “agonizing activity of GAVE19” refer to GAVE19-responsive cells measured in vivo or in vitro based on standard techniques. By an activity affected by a GAVE19 protein, polypeptide or nucleic acid molecule is meant. The GAVE19 activity may be a direct activity such as associated with the enzymatic activity of the second protein or an indirect activity such as a cell signaling activity mediated by the interaction of the GAVE19 protein and the second protein. In particular embodiments, GAVE19 activity includes, but is not limited to, at least one of the following activities: (i) ability to interact with a protein in the GAVE19 signaling pathway; (ii) GAVE19 ligand Ability to interact with; and (iii) ability to interact with intracellular target proteins.

Furthermore, in accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques in the state of the art can be employed. Such techniques are explained fully in the literature. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second 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 ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization [BD Hames & SJ Higgins eds. (1985)]; Transcription And Translation [BD Hames & SJ Higgins, eds. (1984)]; Animal Cell Culture [RI Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausbel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
Therefore, as expressed herein, the following terms have the definitions presented below.

A “vector” refers to a replicon, such as a plasmid, phage or cosmid, to which other DNA segments can be ligated, resulting in replication of the ligated segments. Here, “replicon” means DN in vivo
A autonomous unit of replication, that is, any genetic element (eg, plasmid, chromosome, virus) that has a replicable action under its own control. Specific examples of vectors are described below.

  By “cassette” is meant a DNA segment that can be inserted into a vector at a specific restriction site. This DNA segment 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 said to be “transfected” when exogenous or heterologous DNA is introduced inside the cell. A cell is said to be “transformed” by exogenous or heterologous DNA when the transfected DNA affects phenotypic changes. Preferably, the transforming DNA should be integrated into the chromosomal DNA (covalently linked) and finished into the genome of the cell.

  “Heterologous” DNA refers to DNA that is not naturally present in the cell or present in the chromosome of the cell. Preferably, the heterologous DNA contains a gene that is foreign to the cell.

  “Homologous recombination” refers to insertion of a foreign DNA sequence of a vector into a chromosome. In particular, vectors target specific sites on the chromosome for homologous recombination. For specific homologous recombination, the vector must contain a sufficiently long region that is homologous to the chromosomal sequence for complementary binding and incorporation of the vector into the chromosome. The longer the region with homology, and the greater the degree of sequence homology, the greater the efficiency of homologous recombination.

In one aspect of the isolated nucleic acid molecule according to the present invention, the present invention extends to 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. It is.

  “Nucleic acid molecule” means a polymer of a phosphate ester of ribonucleotide (adenosine, guanosine, uridine or cytidine; “RNA molecule”) or deoxyribonucleotide (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; “DNA molecule”). Refers to phosphoester analogs such as types, or their phosphorothioates and thioesters, and may be either single-stranded or double-stranded helix. Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helix may also be used. The term nucleic acid molecule, in particular DNA or RNA molecule, refers only to the primary and secondary structure of these molecules, and is not limited to any particular tertiary structure. Thus, this term includes double-stranded DNA, especially linear or circular DNA molecules (eg, restriction fragments), plasmids, and chromosomes. Discussing the special double-stranded DNA molecular structure, the sequence is only here as a sequence in the 5 'to 3' direction along the non-transcribed DNA strand (ie a strand having a sequence homologous to the mRNA). It is written according to the usual conventions given. “Recombinant DNA molecule” means a DNA molecule that has undergone a molecular biological manipulation.

  An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source material of the nucleic acid. In particular, an “isolated” nucleic acid is a sequence that is naturally present at both ends of the nucleic acid encoding GAVE19 in the genomic DNA of the organ of nucleic acid origin (ie, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). Not included. In some embodiments, an isolated GAVE19 nucleic acid molecule is about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, .0 of nucleotide sequence that is naturally present at both ends of the nucleic acid molecule in the genomic DNA of a cell of nucleic acid origin. It can contain less than 5 kb or 0.1 kb. In addition, “isolated” nucleic acid molecules, such as cDNA molecules, are substantially free of other cellular components or media when made by recombinant techniques, or chemically when made by chemical synthesis. Means substantially free of precursors or other compounds.

Nucleic acid molecules according to the present invention, such as the nucleotide sequence of SEQ ID NO: 1, or any fragment or complement of that nucleotide sequence, or analogs or derivatives thereof, use standard molecular biology techniques and the sequence information provided herein. Can be isolated. By using all or part of the nucleic acid sequence of SEQ ID NO: 1 as a hybrid probe, the GAVE19 nucleic acid molecule can be isolated by standard hybrid and cloning techniques (eg, as described by Sambrook et al.).

  The nucleic acid molecules of the invention can be amplified according to standard PCR amplification techniques using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers. Such a primer can be easily prepared using information obtained from SEQ ID NO: 1 and conventional experimental techniques. Furthermore, oligonucleotides corresponding to the GAVE19 nucleotide sequence can also be prepared using standard synthetic techniques, such as an automated DNA synthesizer.

An isolated nucleic acid molecule capable of hybridizing to GAVE19 DNA The present invention is further capable of hybridizing to a hybrid probe complementary to GAVE19 DNA under stringent hybrid conditions, or hybridizable to GAVE19 DNA, or stringent. It extends to isolated nucleic acid molecules that can hybridize to each other under hybrid conditions. In particular, the present invention relates to an isolated nucleic acid molecule capable of hybridizing under stringent hybrid conditions with a nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1, or an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1. It extends to probes that are complementary to nucleic acid molecules.

  A nucleic acid molecule can be annealed with other nucleic acid molecules under conditions of appropriate temperature and solution ionic strength (see Sambrook et al., Below) conditions, such as cDNA, genomic DNA, or RNA. It is said to be “hybridizable” with other nucleic acid molecules. The conditions of temperature and ionic strength determine the “stringency” of the hybrid.

For primary screening of homologous nucleic acids, low stringency hybrid conditions corresponding to a T _{m of} 55 ° C. can be used (eg, 5 × SSC, 0.1% SDS,
0.25% milk, no formamide added; or 30% formamide, 5 × SSC, 0.5% SDS). As moderate stringency hybrid conditions corresponding to higher T _m , for example, 40% formamide, 5 × or 6 × SSC can be used. As high stringency hybrid conditions corresponding to the highest T _m , for example, 50% formamide, 5 × or 6 × SSC can be used.

Hybrids require that two nucleic acids contain complementary sequences, but depending on the stringency of the hybrid, mismatches between bases can also occur. It is well-known in the art that the appropriate stringency for nucleic acid hybrids can vary depending on the length of the nucleic acids and the degree of complementarity. The higher the degree of similarity or homology between two nucleotide sequences, the higher the _Tm value of a hybrid of nucleic acids having those sequences. The relative stability of nucleic acids (corresponding to higher T _m ) decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids longer than 100 nucleotides in length, an equilibrium constant for calculating _Tm has been derived (see Sambrook et al., 9.50-0.51 below). For hybrids using shorter nucleic acids or oligonucleotides, the position of the mismatch becomes more important and the length of the oligonucleotide will determine its specificity (see Sambrook et al., 11.7-11. 8). The shortest length of a hybridizable nucleic acid molecule is at least about 20 nucleotides, preferably at least about 30 nucleotides, more preferably at least about 40 nucleotides, even more preferably at least about 50 nucleotides, and most preferably at least about 60 nucleotides. In a particular embodiment of the invention, the hybridizable nucleic acid molecule is at least 300, 325, 350, 375, 400, 450, 500, 600, 650, 700, 800, 900, 1000 or 1100 nucleotides in length; And hybridizing under stringent conditions with a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, its complement or a fragment thereof, preferably the coding sequence.

As used herein, the term “hybridizes under stringent conditions” means that the nucleotide sequence is at least 55%, 60%, 65%, 70%, and preferably 75%, or more complementary. It is meant to describe the hybrid and wash conditions that typically maintain the hybrid with each other. Such stringency conditions are known in the art and can be found in “Current Protocols in Molecular Biology”, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A preferred but non-limiting example of stringent hybrid conditions is that it hybridizes at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC) and 50 × 0.2 × SSC in 0.1% SDS. Washing once or more at ˜65 ° C. may be mentioned. Preferably, an isolated nucleic acid molecule of the present invention that hybridizes to the sequence of SEQ ID NO: 1 under stringent conditions, or a complement thereof, can be cited corresponding to a naturally occurring nucleic acid molecule. As used herein, a “naturally occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (eg, one that encodes a natural protein). One skilled in the art will appreciate that the conditions can be varied in terms of sequence specific variability (eg, length, GC content, etc.).

  The present invention is intended to encompass nucleic acid fragments of GAVE19 that can be used as diagnostics for GAVE19 analogs with similar properties. This diagnostic fragment may be derived from any part of the GAVE19 gene containing sequences at both ends. This fragment can be used as a library probe to practice known methods.

  Furthermore, the nucleic acid molecules of the present invention also include those that are only part of the nucleic acid sequence encoding GAVE19, such as fragments that can be used as probes or primers, and fragments that encode biologically active portions of GAVE19. It is a waste. 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.

  Nucleotide sequences determined by cloning of the human GAVE19 gene create probes and primers for identification and / or cloning of GAVE19 homologues of other cell types, eg, other tissues and GAVE19 homologues from other animals be able to. This probe / primer typically contains a fully purified oligonucleotide. The oligonucleotide is at least about 12, preferably 25, more preferably 50, 75, 100, 125, 150, 175, 200 of the sense or antisense sequence of SEQ ID NO: 1, or a naturally occurring mutant of SEQ ID NO: 1. , 250, 300, 350, or 400 contiguous nucleotides typically contain regions of nucleotide sequence that hybridize under stringent conditions. Probes based on the GAVE19 nucleotide sequence can be used to assay transcripts or genomic sequences encoding similar or identical proteins.

  As used herein, the term “fragment” or “portion” of an isolated nucleic acid molecule according to the present invention is at least 12, preferably about 25, more preferably about 50, 75, 100, 125. , 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides. Hence, a “fragment” of an isolated nucleic acid molecule according to the present invention is not simply 1 or 2 nucleotides.

Similarly, a “fragment” or “portion” of a polypeptide according to the invention contains at least 9 consecutive amino acid residues. A particular example of a polypeptide fragment according to the invention comprises an epitope that binds to the GAVE19 antibody, or fragment thereof.

A nucleic acid fragment encoding "a biologically active portion of GAVE19" is a method of isolating a portion of SEQ ID NO: 1 encoding a polypeptide having GAVE19 biological activity, expressing a encoded portion of a GAVE19 protein (Eg, in vitro recombinant expression) and measuring the activity of the encoded portion of GAVE19.
The present invention further includes a nucleic acid molecule that differs from the nucleotide sequence of SEQ ID NO: 1 due to the degeneracy of the genetic code and therefore encodes a GAVE19 protein similar to that encoded by the nucleotide sequence set forth in SEQ ID NO: 1.

Homologous Nucleic Acid Molecules The present invention further extends to isolated nucleic acid molecules that are homologous to the GAVE19 DNA molecule, such as those that are homologous to the isolated nucleic acid molecule having the DNA sequence of SEQ ID NO: 1. When at least about 50% (preferably at least about 75%, and most preferably at least about 90% or 95%) of the nucleotides of a DNA sequence with a specified length make a pair, the two DNA sequences are “ “Substantially homologous” or “substantially similar”.

Sequences that are substantially homologous can be obtained using a sequence data bank using default parameters, or standard software that can be used, for example, in Southern hybridization experiments under stringent conditions defined for specialized systems. It can be identified by comparing the sequences. The determination of suitable hybridization conditions is within the state of the art. For example, see Maniatis et al. Described later; DNA Cloning Vols. I & II described later; Nucleic Acid Hybridization described later.
Furthermore, nucleic acid molecules encoding GAVE19 proteins from other species (GAVE19 homologues) using nucleotide sequences different from human GAVE19 are also intended to be within the scope of the present invention.

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

Nucleic acid molecules corresponding to natural allelic variants and homologues of GAVE19 cDNA according to the present invention are obtained here using mouse cDNA or a portion thereof as a hybridization probe according to standard hybrid technology under stringent hybrid conditions. Isolation can be based on the identity of the disclosed murine GAVE19 nucleic acid.

  As used herein, the term “corresponds” refers to sequences that are similar or homologous, regardless of whether the exact position is the same or different from the molecule for which similarity or homology was measured. Thus, the term “corresponds” refers to sequence similarity and not the number of amino acid residues or nucleotide bases.

  Furthermore, due to the degenerate nature of the codons in the genetic code, the GAVE19 protein according to the present invention is encoded by a number of isolated nucleic acid molecules. “Degenerate property” means the use of a different three letter codon that identifies a particular amino acid according to the genetic code. It is well known in the art that the following codons are used interchangeably for each specific amino acid code.

Phenylalanine (Phe or F): UUU or UUC
Leucine (Leu or L): UUA or UUG or CUU or CUC or CUA or CUG
Isoleucine (Ile or I): AUU or AUC or AUA
Methionine (Met or M): AUG
Valine (Val or V): GUU or GUC or GUA or GUG
Serine (Ser or S): UCU or UCC or UCA or UCG or AGU or AGC
Proline (Pro or P): CCU or CCC or CCA or CCG
Threonine (Thr or T): ACU or ACC or ACA or ACG
Alanine (Ala or A): GCU or GCG or GCA or GCG
Tyrosine (Tyr or Y): UAU or UAC
Histidine (His or H): CAU or CAC
Glutamine (Gln or Q): CAA or CAG
Asparagine (Asn or N): AAU or AAC
Lysine (Lys or K): AAA or AAG
Aspartic acid (Asp or D): GAU or GAC
Glutamic acid (Glu or E): GAA or GAG
Cysteine (Cys or C): UGU or UGC
Arginine (Arg or R): CGU or CGC or CGA or CGG or AGA or AGG
Glycine (Gly or G): GGU or GGC or GGA or GGG
Tryptophan (Trp or W): UGG

Stop codon UAA (Ocher), UAG (Amber) or UGA (Opal)
The codons specified above are for RNA sequences. The corresponding DNA codon is one in which U is replaced with T.

  It will be appreciated by those skilled in the art that in addition to the murine GAVE19 nucleotide sequence found in SEQ ID NO: 1, DNA sequence polymorphisms that cause changes in the amino acid sequence of GAVE19 may exist in a population. Seem. Such genetic polymorphism in the GAVE19 gene may also exist within an individual of a population due to natural allelic variation. An allele refers to one of a group of genes that occur selectively at the same locus. As used herein, the terms “gene” and “recombinant gene” refer to a nucleic acid molecule comprising an open reading frame encoding an GAVE19 protein, preferably a mammalian GAVE19 protein. As used herein, the phrase “allelic variant” refers to a nucleotide sequence that occurs at the GAVE19 locus or a polypeptide encoded by a nucleotide sequence. Selective alleles can be identified by sequence analysis of genes of interest in a number of different individuals. It can easily be done using hybridization probes to identify the same locus in different individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms, as well as variations of GAVE19 resulting from natural allelic variation, that do not alter the functional activity of GAVE19, are within the scope of the present invention. is there.

  In addition, variants of the isolated nucleic acid molecules according to the present invention can be readily prepared by one of the state of the art using routine experimental techniques such as site-directed mutagenesis.

Antisense Nucleotide Sequences The present invention is also complementary to antisense nucleic acid molecules, ie, sense nucleic acids encoding proteins, eg, complementary to the coding strand of a double stranded cDNA molecule or complementary to an mRNA sequence. It extends to molecules that are. Therefore, an antisense nucleic acid can bind to a sense nucleic acid by hydrogen bonding. An antisense nucleic acid is complementary to the entire GAVE19 coding strand or only a portion thereof, eg, all or a portion of a protein coding region (ie, open reading frame). An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding GAVE19. Non-coding regions ("5 'and 3' untranslated regions") are 5 'and 3' sequences that flank the coding region and are not translated into amino acids.

Regarding the coding strand sequence (eg, SEQ ID NO: 1) encoding GAVE19 disclosed herein, the antisense nucleic acid according to the present invention can be designed according to the rules of Watson-Crick base pairing. An antisense nucleic acid molecule can be complementary to the entire coding region of GAVE19 mRNA, but more preferably is an oligonucleotide that is antisense only to a portion of the coding or non-coding strand of GAVE19 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GAVE19 mRNA. Antisense oligonucleotides are, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. The antisense nucleic acid according to the present invention can be constituted by chemical synthesis and enzymatic ligation reaction using processes known in the art. For example, an antisense nucleic acid (eg, an antisense oligonucleotide) is a natural nucleotide or increases the physical stability of a duplex structure formed to increase biological activity or between an antisense and a sense nucleic acid. Therefore, it can be chemically synthesized using various nucleotides chemically modified using, for example, phosphorothioate derivatives, phosphonate derivatives and acridine substituted nucleotides.

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-galactosylquaosin, inosine, N ⁶ -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2 -Methylguanine, 3-methylcytosine, 5-methylcytosine, N ⁶ -adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylcuocosine, 5 -Methoxycal Carboxymethyl uracil, 5-methoxy uracil, 2-methylthio--N ⁶ - isopentenyladenine, uracil-5-oxy acetic acid, wybutosine,
Pseudouracil, cuocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2 -Thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.
Alternatively, an antisense nucleic acid is obtained using an expression vector in which the nucleic acid is subcloned into an antisense orientation (ie, RNA transcribed from the inserted nucleic acid is in the antisense orientation relative to the target nucleic acid of interest). It can also be created biologically.

An antisense nucleic acid according to the present invention is typically administered to a subject or generated in situ , so that it hybridizes or binds to cytoplasmic mRNA and / or genomic DNA encoding GAVE19 protein. Thereby inhibiting expression of the protein by inhibiting transcription and / or translation. This hybridization forms a stable duplex by conventional nucleotide complementarity or, for example, in the case of an antisense nucleic acid molecule that binds to a DNA duplex, in the major groove of the duplex or in the control of GAVE19 A specific interaction with the region forms a stable double structure.

  An example of the route of administration of antisense nucleic acid molecules according to the present invention is direct injection into a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and administered systemically. For example, for systemic administration, it specifically binds to a receptor or antigen expressed on the selected cell surface by, for example, linking an antisense nucleic acid molecule to a peptide or antibody that binds to the cell surface receptor or antigen. The antisense molecule can be modified to do so.

Antisense nucleic acid molecules can also be delivered into cells using the vectors described herein. In order to achieve a sufficient intracellular concentration of the antisense molecule, it is preferred that the antisense nucleic acid molecule be placed in the vector under the control of a strong pol II or pol III promoter.

  The antisense nucleic acid molecule according to the present invention can be an α-anomeric nucleic acid molecule. α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA, where the 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: 327- 330).

Ribozyme (RNA enzyme)
The invention also extends to ribozymes (RNA enzymes). A ribozyme is a catalytic RNA molecule having ribonuclease activity, and can cleave a single-stranded nucleic acid such as mRNA that hybridizes to a ribozyme. Thus, ribozymes (eg, hammerhead ribozymes (described in Haselhoff et al., Nature (1988) 334: 585-591)) can be used to catalytically cleave GAVE19 mRNA transcripts and GAVE19 A ribozyme having specificity for a nucleic acid encoding GAVE19 can be designed based on the nucleotide sequence of GAVE19 DNA disclosed herein (eg, SEQ ID NO: 1). For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed such that the nucleotide sequence of its active site is complementary to the nucleotide sequence that is cleaved into mRNA encoding GAVE19. US Patent Nos. 4,987,071 and 5,116,742). Alternatively, GAVE19 mRNA can be used to select a catalytic RNA having specific ribonuclease activity from a pool of RNA molecules (see, eg, Bartel et al., Science (1993) 261: 1411-1418).

Triple Helix Nucleic Acid Molecules and Peptide Nucleic Acids of the Invention The present invention also extends to nucleic acid molecules that form triple helix structures. For example, GAVE19 gene expression is blocked by a target nucleotide sequence complementary to the control region of GAVE19 (eg, GAVE19 promoter and / or enhancer), forming a triple helix structure that blocks transcription of the GAVE19 gene in target cells (reviews). Helene, Anticancer Drug Des (1991) 6 (6): 569; Helene Ann NY Acad Sci (1992) 660: 27; and Maher, Bioassays (1992) 14 (12): 807).

  As a particular example, the nucleic acid molecules of the invention can be modified at the base residue, sugar residue or phosphate backbone, for example to improve the stability, hybridization or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid can be modified to yield a peptide nucleic acid (Hyrup et al., Bioorgamic & Medicinal Chemistry (1996) 4: 5). As used herein, the term “peptide nucleic acid” or “PNA” means nucleic acid mimics, for example, in the case of DNA mimics, the phosphate backbone of deoxyribose is replaced by a pseudopeptide backbone. It is substituted and retains only four natural nucleobases. The neutral backbone of this PNA has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers was performed using the standard solid phase peptide synthesis protocol described in Hyrup et al., (1996); Perry-O'Keefe et al., Proc Natl Acad Sci USA (1996) 93: 14670, described below. Can be implemented.

  The GAVE19 PNA can also be used for therapeutic or diagnostic applications. For example, PNA can be used as an antisense or anti-gene reagent for sequence-specific regulation of gene expression, for example, by inducing transcriptional or translational inhibition, or by inhibiting replication. GAVE19 PNA can also be used. For example, PNA can be used for analysis of single base pair mutations in genes, eg, in a PNA directed PCR clamp method, in combination with other enzymes, eg, S1 nuclease (Hyrup et al. (1996), described below). When used, it can be used as an artificial restriction enzyme or as a probe or primer in DNA sequencing and hybridization (Hyrup et al. (1996) described below; Perry-O'Keefe et al. (1996) described below. ).

  In other examples, the PAVE of GAVE19 can be modified, for example, to increase stability, specificity or cellular uptake, but it can be modified by attaching a lipophilic or other useful group to the PNA- This can be done by forming a DNA chimera or using other techniques of existing liposomes or drug delivery. The synthesis of PNA-DNA chimeras can be performed as described in the following literature (Hyrup et al. (1996), Finn et al., Nucleic Acids Res (1996) 24 (17): 3357 described below. -63, Mag et al., Nucleic Acids Res (1989) 17: 5973; and Peterser et al., Bioorganic Med Chem Lett (1975) 5: 1119).

GAVE19 protein The invention further extends to 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 GAVE19 protein having a sequence different from SEQ ID NO: 2, for example a variant thereof, introduces one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1. As a result, one or more amino acid substitutions, additions or deletions can be introduced into the protein to be encoded.

In particular embodiments, mutants of the GAVE19 protein are (1) capable of producing protein-protein interactions with proteins in the GAVE19 signaling pathway, (2) ability to bind GAVE19 ligand, or (3) intracellular. It can be used to assay the ability to bind to a target protein.
In yet another example, the GAVE19 mutant can be used to assay the ability to modulate cell proliferation or cell differentiation.

The native GAVE19 protein can be isolated from cell or tissue material by an appropriate purification procedure using standard protein purification techniques. Alternatively, GAVE19 protein can be readily produced by recombinant DNA technology. As another measure according to the present invention, a GAVE19 protein or polypeptide can be chemically synthesized using a standard peptide synthesis technique.

  An “isolated” or “purified” protein, or biologically active portion thereof, substantially comprises contaminant material derived from intracellular material or cells or tissue sources of GAVE19 protein origin. Or in the case of chemical synthesis, is substantially free of synthetic precursors or other chemicals. As used herein, the phrase “substantially free of intracellular material” includes the preparation of GAVE19 protein that is free of cellular components of cells from which the protein has been isolated or produced by recombinant techniques. Thus, GAVE19 protein substantially free of intracellular substances is about 30%, 20%, 10% or 5% or less of non-GAVE protein (also referred to herein as “contaminated protein”), or Also included is the preparation of GAVE19 protein having less (as dry weight).

  If the GAVE19 protein or biologically active portion thereof is produced recombinantly, it is preferably substantially free of medium, ie about 20%, 10% or 5% of the volume of the protein preparation. It means that only the following or less medium is included. When the GAVE19 protein is made by chemical synthesis, it is preferably substantially free of synthetic precursors or other chemicals, i.e. separated from synthetic precursors or other chemicals involved in protein synthesis. Means that Therefore, such GAVE19 protein preparations contain only about 30%, 20%, 10% or 5% or less (as dry weight) of synthetic precursors or non-GAVE19 chemicals.

  A biologically active portion or fragment of the GAVE19 protein is a peptide comprising an amino acid sequence that is sufficiently identical to or derived from the amino acid sequence of the GAVE19 protein (eg, the amino acid sequence represented by SEQ ID NO: 2) And contains fewer amino acids than the full length of the GAVE19 protein and represents at least one activity of the GAVE19 protein. Typically, the biologically active portion includes a domain or motif for at least one activity of the GAVE19 protein. The biologically active portion of the GAVE19 protein can be a polypeptide consisting of, for example, 10, 25, 50, 100 or more amino acids in length. Certain biologically active polypeptides contain one or more identified GAVE19 structural domains.

In addition, portions of other biologically active GAVE19 proteins, in which other protein regions are deleted, can be prepared by recombinant techniques and can also be functionalized with raw GAVE19 protein. One or more of the activities can be assessed.
Another useful GAVE19 protein is substantially the same as SEQ ID NO: 2, and the functional activity of the GAVE19 protein of SEQ ID NO: 2 even if there is a difference in amino acid sequence due to natural allelic variation or mutation Is maintaining. For example, such GAVE19 proteins and polypeptides have at least one of the biological activities described herein.

  Therefore, a useful GAVE19 protein has at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2. It is a protein that contains the amino acid sequence that has the functional activity of the GAVE19 protein of SEQ ID NO: 2. In a specific example, the GAVE19 protein retains the functional activity of the GAVE19 protein having SEQ ID NO: 2.

  In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences need to be aligned to suit the purpose of the optimal comparison (eg, obtaining an optimal alignment with the second amino acid or nucleic acid sequence) In order to introduce a gap in the sequence of the first amino acid or nucleic acid). The amino acid residues or nucleotides corresponding to the amino acid configuration or nucleic acid configuration are then compared. Corresponding to the arrangement of the second sequence, when the arrangement of the first sequence is occupied by the same amino acid residue or nucleotide, the molecules are considered identical in that arrangement. The percent identity of two sequences is a function of the number of configurations with identity occupied by the sequences (ie, percent identity = number of configurations with identity / total number of configurations (eg, overlapping configurations) ) × 100).

In one embodiment, the two sequences are the same length. Determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Specific examples of mathematical algorithms used to compare two sequences include, but are not limited to: Karlin et al., Proc Natl Acad Sci USA (1990) 87: 2264, and Karlin
Et al., Proc Natl Acad Sci USA (1993) 90: 5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul et al., J Mol Bio (1990) 215: 403). To obtain a nucleotide sequence homologous to the GAVE19 nucleic acid molecule of the present invention, the BLAST nucleotide search can be operated using the NBLAST program, for example, setting score = 100, word length = 12. In order to obtain an amino acid sequence homologous to the GAVE19 protein molecule of the present invention, the BLAST protein search can be operated using the XBLAST program with a score = 50 and a word length = 3. To determine the alignment of gaps for comparative purposes, Gapped BLAST can be manipulated as described by Altschul et al. (Altschul et al., Nucleic Acids Res (1997) 25: 3389). Alternatively, PSI-Blast can be manipulated as an iterative search to search for faint interactions between two molecules (Altschul et al. (1997) above). When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used (see http://www.ncbi.nlm.nih.gov.). ).

Another special example of a mathematical algorithm used for sequence comparison is the Myers et al. Algorithm (Mayers et al., CABIOS (1988) 4: 11-17), but is not limited to this. is not. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG array and software package. When using the ALIGN program for comparing amino acid sequences, the PAM120 weight residue table, gap length penalty 12 and gap length penalty 4 can be used.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps. In calculating percent identity, only exact matches should be taken into account.

  The present invention also extends to GAVE19 chimeric or fusion proteins. As used herein, a GAVE19 “chimeric protein” or “fusion protein” is one that includes a GAVE19 polypeptide operably linked to a non-GAVE19 polypeptide. “GAVE19 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to GAVE19. “Non-GAVE19 polypeptide” refers to a protein that is not essentially identical to the GAVE19 protein, eg, a polypeptide that is derived from the same or different organism and has an amino acid sequence that corresponds to a protein that is different from the GAVE19 protein. For the GAVE19 fusion protein, the GAVE19 polypeptide can correspond to all or part of the GAVE19 protein, and preferably can correspond to at least one biologically active portion of the GAVE19 protein.

With regard to the GAVE19 fusion protein, the GAVE19 polypeptide can correspond to all or part of the GAVE19 protein, and preferably can correspond to at least one biologically active portion of the GAVE19 protein. With respect to fusion proteins, the term “operably linked” means that a GAVE19 polypeptide and a non-GAVE19 polypeptide are fused together in frame. The non-GAVE19 polypeptide can be fused to the N-terminus or C-terminus of the GAVE19 polypeptide. One useful fusion protein is GST-GAVE19 in which the GAVE19 sequence is fused to the C-terminus of glutathione-S-transferase (GST). Such fusion proteins facilitate the purification of recombinant GAVE19.

In another embodiment, the fusion protein according to the invention extends to a GAVE19-immunoglobulin fusion protein in which all or part of GAVE19 is fused to a sequence derived from one member of the immunoglobulin protein family. The GAVE19-immunoglobulin fusion protein according to the present invention is mixed in a pharmaceutical composition to inhibit the interaction between GAVE19 ligand and GAVE19 protein on the cell surface, thereby suppressing GAVE19-mediated signal conversion in vivo . Can be administered to the subject. The GAVE19-immunoglobulin fusion protein can be used to affect the available amount of a ligand that is cognate with GAVE19. Inhibition of the GAVE19 ligand-GAVE19 interaction is therapeutically useful for both treating proliferation and differentiation disorders and modulating (eg, promoting or preventing) cell survival. Furthermore, the GAVE19-immunoglobulin fusion protein according to the present invention is used as an immunogen for the production of anti-GAVE19 antibody in a subject, for purification of GAVE19 ligand, and for screening assay for molecular identification that inhibits the interaction between GAVE19 and GAVE19 ligand. Can be used.

  In a special embodiment, the GAVE19 chimera or fusion protein according to the present invention can be made by standard recombinant techniques. For example, DNA fragments encoding different polypeptide sequences can be obtained using conventional techniques, such as the use of blunt or sticky ends for ligation, digestion with restriction enzymes to provide appropriate ends, and sticky ends as appropriate. Can be linked in-frame according to embedment, alkaline phosphatase treatment to avoid improper binding, and enzymatic ligation. In other embodiments, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.

  Alternatively, PCR amplification of gene fragments can be accomplished with anchor primers that create a chimeric gene sequence by creating a complementary overhang between two coupled gene fragments, followed by annealing and reamplification. Can be performed (see eg Ausubel et al., Supra).

  In addition, many expression vectors are commercially available that already encode a fusion target (eg, GST polypeptides). The GAVE19 encoded nucleic acid can be cloned into such an expression vector and the fusion target is linked in frame to the GAVE19 protein.

Variants As explained above, the present invention also extends to variants of the GAVE19 protein. For example, mutations can be introduced into the amino acid sequence of SEQ ID NO: 2 using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. In addition, conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. “Conservative amino acid substitution” refers to an amino acid residue substituted with an amino acid residue having a similar side chain. For example, one or more amino acids are replaced with amino acids having similar polarity and act as a functional equivalent, resulting in a silent mutation (silence mutation). The amino acid substitution in the amino acid sequence of the polypeptide of the present invention can be selected from other amino acids in 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 an aromatic ring structure 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 a conversion does not appear to affect the apparent molecular weight as measured by polyacrylamide gel electrophoresis or isoelectric point.

Particularly preferred substitutions are as follows:
-Arg is replaced by Lys or vice versa, positive charge is maintained;
-Asp is replaced with Glu, or vice versa, the negative charge is maintained;
- replacing Thr by Ser, free -OH is maintained; and - replacing Asn with Gln, the free NH ₂ is maintained.

  Amino acid substitution can also be introduced to replace amino acids having particularly favorable properties. For example, Cys can be introduced at a site that may form a disulfide bridge with another Cys. His can in particular be introduced at the “catalyst” site (ie, His can act as an acid or base and is the most commonly used amino acid as a biochemical catalyst). Pro can be introduced specifically to induce β-turns in protein structures due to its planar structure.

  Mutations can also be induced randomly along all or part of the GAVE19 coding sequence, for example by saturation mutagenesis, and the resulting mutant maintains its activity for GAVE19 biological activity. Screening can be performed to identify mutants. Following mutagenesis, the encoded protein can be expressed by recombinant techniques and the activity of the protein can be measured.

Variants according to the present invention are GAVE19 agonists (mimetic) or GAV
It can act as an E19 antagonist. Variants of the GAVE19 protein can also be generated by mutation, ie individual point mutations or truncations of the GAVE19 protein. An agonist of the GAVE19 protein can substantially maintain the same or a subset of the biological activity of the naturally occurring GAVE19 protein. For example, an antagonist of the GAVE19 protein can competitively bind to the downstream or upstream pathway of an intracellular signaling cascade that includes the GAVE19 protein, and one or more of the naturally occurring activities of the GAVE19 protein. Can be inhibited. In this way, specific biological effects can be elicited by processing variants with limited functions. Treatment of a subject with a variant having a naturally occurring subset of the biological activity of the GAVE19 protein provides the subject with fewer side effects compared to treatment with the naturally occurring form of the GAVE19 protein.

Variants of the GAVE19 protein having the action of either a GAVE19 agonist (mimetic) or a GAVE19 antagonist are mutant screening combinatorial libraries, such as the GAVE19 protein truncation mutant for GAVE19 agonist or antagonist activity. Can be identified. In some embodiments, a versatile library of GAVE19 variants can be generated by combinatorial mutagenesis at the nucleic acid level and can be encoded by a versatile gene library. A versatile library of GAVE19 variants is, for example, a synthetic oligonucleotide mixture with a degenerate set of potential GAVE19 sequences in each polypeptide, or also a larger fusion protein containing a set of GAVE19 sequences therein (eg, (Phage display) can be prepared by enzymatic ligation to gene sequences expressed as a set.

  There are a variety of methods used to generate a library of potential GAVE19 variants from a degenerate oligonucleotide sequence. Chemical synthesis of degenerate gene sequences can be made by synthesizing in an automated DNA synthesizer and then linking the synthesized gene to an appropriate expression vector. By using a degenerate set of genes, all of the sequences encoding the desired set of potential GAVE19 sequences can be prepared as a mixture. Methods for degenerate oligonucleotide synthesis are known in the state of the art (eg, Narang, Tetrahedron (1983) 39: 3; Itakura et al., Ann Rev Biochem (1984) 53: 323; Itakura et al., Science (1984 198: 1056; see Ike et al., Nucleic Acid Res (1983) 11: 477).

  In addition, a library of GAVE19 protein coding sequence fragments can be used to generate a heterogeneous population of GAVE19 fragments for use in screening and subsequent selection of GAVE19 protein variants. In one embodiment, the library encoding the sequence fragment is treated with a nuclease under conditions such that about one nick (break) occurs per molecule of a double-stranded PCR fragment of the GAVE19 coding sequence. After denaturing the strand DNA, regenerate the DNA to form a double stranded DNA containing sense / antisense pairs from different nicks and remove the single stranded portion from the regenerated duplex by S1 nuclease treatment The fragment library thus obtained can be prepared by linking to an expression vector. By this method, the expression library can be derived from an expression library that encodes N-terminal and intracellular fragments of various sizes of the GAVE19 protein.

  Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncations and for screening cDNA libraries of gene products with selected properties . Such techniques can be applied to the rapid screening of gene libraries created by combinatorial mutagenesis of GAVE19 protein. The most widely used technique that can be used for high-throughput analysis of large gene library screening is to clone the gene library into a replicable expression vector, and to transfer the vector containing the library to an appropriate cell. And the expression of the combinatorial gene under conditions that facilitate the isolation of the vector encoding the gene (where the product should be detected) Contains.

  Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional mutations in a library, can be used in combination with a screening assay to identify GAVE19 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 GAVE19 Furthermore, the present invention includes derivatives or analogs of GAVE19 made by chemical modification. The GAVE19 protein according to the present invention can be derivatized by binding one or more chemical components to the protein component.

Chemical components for derivatization : The chemical components suitable for derivatization can be selected from among water-soluble polymers, because GAVE19 analogs or derivatives do not precipitate in water-soluble environments such as physiological environments. . In some cases, the polymer is also pharmaceutically acceptable. Those skilled in the art will select promising polymers taking into account whether the polymer / component linkage is used as a drug, and if so desired dosage, circulation time, proteolytic resistance, and others. Can do.

  These requirements for GAVE19 can be confirmed by using the assay presented here. Examples of water-soluble polymers used here include polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6. -Trioxane, ethylene / maleic anhydride copolymer, polyamino acid (which can be either homopolymer or random copolymer), dextran, poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylene However, it is not limited to these. Polyethylene glycol propionaldehyde appears to be advantageous in industrial production due to its stability in water.

  The polymer can be of any molecular weight and can be either branched or linear. In the case of polyethylene glycol, the preferred molecular weight is in the range of about 2 kDa to 100 kDa for ease of handling and production (the term “about” means that in polyethylene glycol synthesis, certain molecules are larger than the stated molecular weight, and Meaning that some molecules are smaller).

Other sizes can be used depending on the desired drug aspect (e.g., desired release time, bioactivity if available, ease of handling, degree of immunogenicity) Or its deficiencies, and known effects of polyethylene glycol on other therapeutic proteins or analogs thereof).
Since many polymer molecules bind to GAVE 19, one skilled in the art would be able to confirm the effect on action. Some molecules are monoderivatives, and some molecules are di-, tri-, tetra-derivatives, or derivatives with combinations of the same or different chemical residues (eg, polymers such as polyethylene glycols of different molecular weights). . The ratio of polymer molecules to GAVE19 molecules depends on the concentration of the reaction mixture. In general, the optimal ratio (as a term for reaction efficiency in the sense that there is no excess unreacted components and polymer) is selected depending on the desired degree of derivatization (eg mono, di-, tri-, etc.) It is determined by factors such as the molecular weight of the polymer, whether the polymer is branched or linear, and the reaction conditions.

Polyethylene glycol molecules (or other chemical components) should bind to GAVE19 taking into account its effect on GAVE19 function or antigenic domain. Several conjugation methods are possible for those skilled in the art, such as those described in the literature in EP 0 401 384 (PEG coupling to G-CSF) and Malik et al., 1992, Exp. Hematol. 20: 1028-1035 (paging of GM-CSF using tresyl chloride has been reported). For example, polyethylene glycol can be covalently linked to an amino acid residue through an active group such as a free amino acid or a carboxy group. Here, the active group is a group to which an activated polyethylene glycol molecule can bind. Amino acid residues having a free amino group include lysine residues and N-terminal amino acid residues, and amino acid residues having a free carboxy group include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues . Sulfhydryl groups (-SH groups) can also be used as active groups for binding polyethylene glycol molecules. For therapeutic purposes, linkage at the amino group, such as at the N-terminus or lysine group, is preferred.

  Specifically, GAVE19 in which the N-terminus is chemically modified is desirable. Using polyethylene glycol as described in this composition, from various polyethylene glycol molecules (by molecular weight, branched chain, etc.), the ratio of polyethylene glycol molecules to GAVE19 molecules in the reaction mixture, the type of PEGylation reaction to be performed And methods of obtaining selective N-terminal pegylated proteins can be selected. The method of obtaining an N-terminal pegylation preparation (ie, if necessary, separating this molecule from other monopegylation molecules) can be performed by purifying the N-terminal pegylation molecule from a population of pegylation protein molecules. . Selective N-terminal chemical modification can be performed by reductive alkylation exploiting the specific reactivity of the primary amino group difference (lysine vs. N-terminus) that is possible for GAVE19 derivatization. Under appropriate reaction conditions, substantially selective derivatization of GAVE19 at the N-terminus with a polymer containing a carbonyl group can be achieved. For example, selective N-terminal pegylation of GAVE19 is performed by reaction at a pH such that the pKa difference between the ε-amino group of a lysine residue and the α-amino group of the N-terminal residue of GAVE19 can be utilized. be able to. Such selective derivatization modulates the binding of the water-soluble polymer to GAVE19: conjugation with the polymer occurs predominantly at the N-terminus of GAVE19 and other active groups such as the amino group of the lysine side chain. Almost no modification occurs. When reductive alkylation is used, the water-soluble polymer falls into the type described above and must have one active aldehyde in order to couple to GAVE19. Polyethylene glycol propionaldehyde can be used because it contains one active aldehyde.

GAVE19 antibody, variant, fragment or analog or derivative thereof Isolated GAVE19 protein or subpart or fragment thereof is used to produce antibodies that bind to GAVE19 using standard techniques of polyclonal or monoclonal antibody preparation. Can be used as an immunogen. As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that specifically binds an antigen, such as GAVE19 or fragments thereof. Say. A molecule that specifically binds to GAVE19 refers to a molecule that binds to GAVE19, but does not substantially bind to other molecules in biological preparations that naturally contain, for example, GAVE19. Examples of immunologically active portions of immunoglobulin molecules include F _(ab) and F _(ab ' _{) 2} fragments obtained by treating an antibody with an enzyme such as pepsin. The present invention provides polyclonal, monoclonal and chimeric antibodies having GAVE19, a variant thereof, a fragment thereof or an analogue or derivative thereof as an immunogen.

  The full length GAVE19 protein can be used, and the present invention instead provides an antigenic peptide fragment of GAVE19 for use as an immunogen. The antigen peptide of GAVE19 contains at least 8 (preferably 10, 15, 20, 30 or more) amino acid residues of the amino acid sequence presented in SEQ ID NO: 2, and forms a specific immune complex with GAVE19 Contains the epitope of GAVE19 that raises antibodies against the peptide.

A GAVE19 immunogen can typically be used to prepare antibodies by immunizing a suitable animal (eg, rabbit, goat, mouse or other mammal) with the immunogen. Suitable immunogenic preparations may include, for example, recombinantly expressed GAVE19 protein or chemically synthesized GAVE19 polypeptide.
The preparation can further include an adjuvant, such as complete or incomplete Freund's adjuvant, or similar immunostimulatory substance. A polyclonal anti-GAVE19 antibody response is elicited by immunizing a suitable animal with a GAVE19 immunogen preparation.
The antibody according to the present invention is a monoclonal antibody, a polyclonal antibody or a chimeric antibody. As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to a population of antibody molecules comprising a single type of antigen binding site capable of immunoreacting with a specific epitope of GAVE19. . Monoclonal antibody compositions thus typically present a single binding affinity for a specific GAVE19 protein epitope.

  Polyclonal anti-GAVE19 antibodies can be prepared by immunizing a suitable animal with the GAVE19 immunogen as described above. The titer of anti-GAVE19 antibody in the immunized animal can be measured over time by standard techniques such as enzyme immunoassay (ELISA) using immobilized GAVE19. If necessary, antibody molecules against GAVE19 can be isolated from mammals (eg, from blood) and further purified by known techniques such as protein A chromatography to obtain an IgG fraction. it can. At an appropriate time after immunization, eg, when the anti-GAVE19 antibody titer reaches a maximum, antibody-producing cells are collected from the subject and hybridoma technology first described by Kohler et al. (Kohler et al. , 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 Antibodies and Cancer Therapy, (1985) ), Alan R. Liss, Inc., pp. 77-96) or by standard techniques such as trioma technology. Techniques for producing hybridomas are well known (for review see Current Protocols in Immunology (1994) Coligan et al., Eds., John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically myeloma) is fused to a mammalian lymphocyte (typically a spleen cell) immunized with a GAVE19 immunogen as described above, and the resulting hybridoma To identify a hybridoma producing a monoclonal antibody that binds to GAVE19.

Any of a number of known protocols used for fusion of lymphocytes and immortalized cell lines can be utilized for the purpose of generating anti-GAVE19 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 Analyses, 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 on such methods, and that they are also useful. Typically, immortalized cell lines (eg, myeloma cell lines) are derived from mammals that are allogeneic to lymphocytes. For example, mouse hybridomas are sensitive to lymphocytes from mice immunized with the immunogen preparation of the present invention against a medium containing immortalized mouse cell lines, such as hypoxanthine, aminopterin and thymidine (HAT medium). It can be created by fusing with myeloma cell line.
Any of a number of myeloma cell lines (eg, P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines) should be used as fusion partners according to standard techniques. Can do. These myeloma lines are available from ATCC. Typically, HAT-sensitive murine eloma cells are fused with murine spleen cells using polyethylene glycol (PEG). Hybridoma cells obtained by fusion can be selected using HAT medium that kills non-fused or non-productive fused cells (unfused splenocytes die after several times because they are not transformed). Hybridoma cells producing a monoclonal antibody according to the present invention can be detected using a screening of the supernatant of a hybridoma culture containing an antibody that binds to GAVE19 (eg, standard ELISA method).

Instead of preparing a hybridoma that secretes a monoclonal antibody, a monoclonal anti-GAVE19 antibody is used to isolate a recombinant combinatorial immunoglobulin library (eg, antibody phage) with GAVE19 to isolate immunoglobulin library members that bind to GAVE19. Display libraries) can be identified and isolated. Phage display library creation and screening kits are commercially available (eg, the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene “SURFZAP” Phage Display Kit, Catalog No. 240612).

  In addition, examples of methods and reagents that can be used specifically for the generation and screening of antibody display libraries include, for example, US Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91 / 17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No WO 90/02809; 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.

  Still further, recombinant anti-GAVE19 antibodies can be made using standard recombinant DNA techniques, including, for example, monoclonal and chimeric antibodies. Such antibodies are known in the art as recombinant DNA technology, such as PCT Publication No. WO 87/02671; Europe Patent Application No. 184,187; Europe Patent Application No. 171,496; Europe Patent Application No. 173,494; PCT Publication No. WO 86/01533; US Patent No. 4,816,567; Europe Patent Application No. 125,023; 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 et al., 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 Using the method described in Beidler et al., J Immunol (1988) 141: 4053-4060 Can be created.

Anti-GAVE19 antibodies (eg, monoclonal antibodies) can be used to isolate GAVE19 by standard techniques such as affinity chromatography or immunoprecipitation. Anti-GAVE19 antibodies can facilitate purification from natural GAVE19 from cells and from host cells that have expressed recombinantly prepared GAVE19.
In addition, anti-GAVE19 antibodies can be used to detect GAVE19 protein (eg, in cell lysates or cell supernatant fractions) to assess the number and pattern of expression of GAVE19 protein. Anti-GAVE19 antibodies can be used as part of a clinical laboratory procedure, for example, in diagnostics to monitor protein levels in tissues to determine the effect of a given treatment. Detection can be facilitated by coupling the antibody to a detectable substance (label) described below.

Detectable labels In some cases, isolated nucleic acid molecules according to the present invention, polypeptides according to the present invention and antibodies according to the present invention, and fragments thereof can be detectably labeled. Suitable labels include enzymes, fluorophores (eg fluorophores such as fluorescent isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide salts, especially Eu ³⁺ ), Mention may be made of chromophores, radioisotopes, chelating agents, dyes, gold colloids, latex particles, ligands (eg biotin), bioluminescent substances, and chemiluminescent reagents.

When employing control markers, the same or different labels can be used for the receptor and control markers. Isotope ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸
When using radioactive labels such as Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re, known and currently available measurement methods can be utilized. If the label is an enzyme, detection can be accomplished by any of the colorimetric, spectroscopic, fluorescent spectroscopic, amperometric or gas quantitative methods known in the state of the art currently in use.

  Direct labeling is one example of a label that can be used in accordance with the present invention. Direct labeling is defined as being readily visible in the natural state with the naked eye or with the aid of an optical filter and / or enhanced utilization such as, for example, ultraviolet light that generates fluorescence. Examples of colored labels that can be used according to the present invention include metal sol particles such as gold sol particles as described in Leuvering (US Patent 4,313,734), Gribnau et al (US Patent 4,373,932) and May et al (WO 88 Dye sol particles, as described in US / 08534), dyed latex as described in May (described above), Snyder (EP-A 0 280 559 and 0 281 327), or liposome as described in Campbell et al. (US Pat. No. 4,703,017). And encapsulated dyes. Other direct labels include radioactive nucleotides, fluorophores, or luminophores. In addition to these direct labeling methods, indirect labels including enzymes can also be used according to the present invention. Various types of enzyme immunoassays are well known in the art, such as alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, and these and others are Eva Engvall, Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and US Patent 4,857,453.

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

In another embodiment, a phosphorylation site is created on an isolated polypeptide according to the invention, an antibody according to the invention or a fragment thereof for labeling with ³² P, European Patent No. issued to Foxwell et al. On October 17, 1995.
0372707 or US Patent No. 5,459,240.

As illustrated herein, proteins including antibodies can be labeled with metabolic labels. Metabolic labeling is performed during in vitro incubation of cells expressing the protein in the presence of a medium supplemented with a metabolic label such as [ ³⁵ S] -methionine or [ ³² P] -orthophosphate. In addition to metabolic (or biosynthetic) labeling using [ ³⁵ S] -methionine, the present invention further includes [ ¹⁴ C] -amino acids and [ ³ H] -amino acids (those not substituted with tritium). The signs used are also taken into account.

Recombinant Expression Vectors and Host Cells Another aspect of the present invention relates to vectors, particularly expression vectors that contain a nucleic acid encoding GAVE19 (or a portion thereof). As explained above, one type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which other DNA fragments can be ligated. Certain vectors are capable of autonomous replication in host cells (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are taken into the host cell, integrated into the host cell's genome, and thus replicated along the host genome. Note that the expression vector can control the expression of the operably linked gene. In general, expression vectors used in recombinant DNA technology are often used in plasmid (vector) form. However, the present invention is intended to include other types of expression vectors such as viral vectors that perform similar functions (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

The recombinant expression vector of the present invention contains a nucleic acid molecule according to the present invention of a type suitable for nucleic acid expression in a host cell. That is, the recombinant expression vector according to the present invention contains one or more control sequences selected on the basis of the host cell used for expression, and it is operably linked to the nucleic acid to be expressed. ing. A recombinant expression vector is “operably linked” when the nucleotide sequence of interest allows the expression of the nucleotide sequence (eg, in an in vitro transcription / translation system or the vector is incorporated into a host cell). When in a host cell) is linked to a regulatory sequence.

  The term “regulatory sequence” is meant to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology Vol. 185, Academic Press, San Diego, CA (1990). Control sequences include direct constitutive expression of nucleotide sequences in many types of host cells (eg, tissue specific control sequences). One skilled in the art will appreciate that the design of the expression vector will depend on factors such as the choice of host cell to transform, the degree of protein expression desired, and the like. The expression vector according to the present invention may be incorporated into a host cell to produce a protein or peptide encoded by the nucleic acid described herein (eg, GAVE19 protein, GAVE19 mutant, fusion protein, etc.). Can do.

The recombinant expression vector according to the present invention is a prokaryotic cell or a eukaryotic cell such as E. coli.
Can be designed to express GAVE19 in bacterial cells, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further discussed in the aforementioned Goeddel. Alternatively, the recombinant expression vector can be transcribed and translated in vitro , for example using phage regulatory elements and proteins such as T7 promoter and / or T7 polymerase.

Protein expression in eukaryotic cells is most often performed in E. coli using vectors containing constitutive or inducible promoters that govern the expression of fused or unfused proteins. A fusion vector adds many amino acids to the protein encoded therein, usually the amino terminus of a recombinant protein. Such fusion vectors typically have three purposes: 1) to increase the expression of the recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to act as a ligand in affinity purification. It plays a role in assisting the purification of the recombinant protein. Often in fusion expression vectors, proteolytic cleavage sites are introduced at the junction of the fusion, allowing separation of the recombinant protein from the fusion and subsequent purification of the fusion protein. Such enzymes and cognate discrimination sequences include factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith et al., Gene (1988) 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ). , They fuse glutathione-5-transferase (GST), maltose E binding protein or protein A, respectively, to the target recombinant protein.

Suitable inducible non-fused E. coli expression vectors include pTrc (Amann et al., Gene (1988) 69: 301-315) and pET lld (Studier et al., Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California (1990) 185: 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.

One strategy to optimize recombinant protein expression in E. coli is to express the protein in a host that lacks the ability to cleave the recombinant protein by proteolysis (Gottesman, Gene Expression Technology: Methods in Enzymolygy, Academic Press, San Diego, California (1990) 185: 119-128). Another alternative is to modify the nucleic acid sequence of the nucleic acid molecule to be inserted into the expression vector so that individual codons for each amino acid are preferably used in E. coli. (Wada et al., Nucleic Acids Res (1992) 20: 2111-2118). Such modifications of the nucleic acid molecules according to the present invention can be made by standard DNA synthesis techniques.

In other embodiments, the GAVE19 expression vector is a yeast expression vector. Examples of vectors for expression in yeast such as baker's yeast ( S. 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). .

Alternatively, GAVE19 can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors that can be used for protein expression in cultured insect cells include the pAc system (Smith et al., Mol Cell Biol (1983) 3: 2156-2165) and
The pVL system (Lucklow et al., Virology (1989) 170: 31-39) is included.

  In still other embodiments, the nucleic acids according to the present invention are also expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors utilized herein include pCDM8 (see Nature (1987) 329: 840) and pMT2PC (Kaufman et al., EMBO J (1987) 6: 187-195), provided that However, it is not limited to these. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are those derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Sambrook et al., References 16 and 17 above.

  In another embodiment, the recombinant mammalian expression vector according to the present invention is suitably used in certain special cell types (eg those in which tissue-specific restriction factors are used for the expression of nucleic acids) It can dominate the expression of nucleic acids. Tissue specific regulators are known in the state of the art. Examples of suitable tissue specific promoters include albumin promoter (liver specificity; Pinkert et al., Genes Dev (1987) 1: 268-277), lymphocyte specific promoter (Calame et al., Adv Immunol ( 1988) 43: 235-275), in particular the T cell receptor promoter (Winoto et al., EMBO J (1989) 8: 729-733) and immunoglobulin (Banerji et al., Cell (1983) 33: 729- 740; Queen et al., Cell (1983) 33: 741-748), neuron-specific promoters (e.g. 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 whey promoter; US Patent No. 4,873,316 and Europe Application No. 264,166). Developmentally regulated promoters are also included, such as the mouse hox promoter (Kessel et al., Science (1990) 249: 374-379) and the alpha-fetoprotein promoter (Campes et al., Genes Dev (1989) 3: 537-546). There is.

The present invention further provides a recombinant expression vector comprising a DNA molecule according to the present invention cloned in an antisense orientation in the expression vector. That is, the DNA molecule is operably linked to a regulatory sequence in a manner that allows expression of an RNA molecule that is antisense to GAVE19 mRNA (by transcription of the DNA molecule). Control sequences operably linked to the nucleic acid cloned in the antisense orientation can be selected to govern the continuous expression of the antisense RNA molecule in a variety of cell types. For example, viral promoters and / or enhancers or control sequences can be selected to govern constitutive tissue-specific or cell type-specific expression of antisense RNA.
Antisense expression vectors are in the form of recombinant plasmids, phagemids or attenuated viruses in which antisense nucleic acids are produced under the control of highly efficient control regions, the activity depending on the cell type into which the vector has been introduced. Can be determined. For a discussion of gene expression control using an antisense gene, reference can be made to Weintraub et al. (Reviews-Trends in Genetics, Vol 1 (1) 1986).

  Another aspect of the present invention relates to a host cell into which a recombinant expression vector according to the present invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms do not simply mean a particular subject cell, but also the progeny of such a cell or its potential progeny. Such progeny may not actually be identical to the parental cell, as some changes may occur during subculture, either due to mutations or environmental influences, but still used here. To be included within the scope of the term.

The host cell can be any prokaryotic or eukaryotic cell. For example, GAVE19 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 using conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to various techniques recognized as a technique for introducing foreign nucleic acid (eg, DNA) into a host cell, such as calcium phosphate or chloride. Calcium coprecipitation, transduction, DEAE-dextran mediated transfection, lipofection, or electroporation are included.

  For stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a few of the cells are known to integrate foreign DNA into the genome. . In order to identify and select this integrant, a gene encoding a selectable marker (eg, conferring resistance to antibiotics) is generally incorporated into the host cells along with the gene of interest. Suitable selectable markers include those that confer drug resistance such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell using the same vector encoding GAVE19 or using other vectors. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene survive, but die to other cells).

  A host cell according to the present invention, such as a prokaryotic or eukaryotic cell in culture, can be used to produce (ie, express) GAVE19 protein. Therefore, the present invention also provides a method for producing GAVE19 protein using the host cell according to the present invention. In one embodiment, the method comprises culturing a host cell according to the present invention (in which a recombinant expression vector encoding GAVE19 has already been introduced) in a stable medium in which GAVE19 protein is produced. including. In another embodiment, the method further comprises isolating GAVE19 from the medium or the host cell.

In another alternative embodiment, GAVE19 contains an inducible expression system for recombinant expression of other proteins subcloned into the modified expression vector. For example, host cells containing a mutated G protein (eg, yeast cells, Y2 adrenal cortex cells and -cyc S49, US Pat. Nos. 6,168,927B1, 5,739,029 and 5,482,835; Mitchell et al., Proc Natl Acad Sci USA (1992) 89 (19): 8933-37 and Katada et al., J Biol Chem (1984) 259 (6): 3586-95) show that GAVE19 is functionally expressed in host cells. Transduced with a vector first expression vector containing a nucleic acid sequence encoding GAVE19. For example, even if the expressed GAVE19 is constitutively active, the mutant does not tolerate signal transduction; that is, activation of the G protein in the downstream cascade direction does not occur (eg, adenylyl cyclase Activation does not occur). Therefore, the second expression vector is used for transduction of GAVE19-containing host cells. The second vector is a structural gene that is complementary to a host cell G protein mutation (ie, a functional mammalian or yeast G _s , G _i , G _o , or G _q , eg, PCT Publication No. WO 97/48820). US Pat. Nos. 6,168,927 B1, 5,739,029 and 5,482,835) in addition to the gene of interest to be expressed by the induction system.
The complementary structural gene of the second vector is inducible, ie complementary under the control of exogenously added components (eg, tetracycline, IPTG, small molecules, etc., see Sambrook et al., Supra). Activates a promoter operably linked to a structural gene. In addition to inducers, proteins encoded by complementary structural genes are functionally expressed, constitutively active GAVE19 forms a complex, and leads to activation of appropriate downstream pathways (eg, , Formation of secondary messenger). The gene of interest, including the secondary vector, has an operably linked promoter activated by an appropriate second messenger (eg, CREB, AP1 factor). Thus, as the second messenger accumulates, the promoter upstream from the gene of interest is activated to express the gene product. In the absence of inducer, the expression of the gene of interest is switched off.

In a special embodiment, the host cell of the inducible expression system includes, but is not limited to, S49 (cyc ⁻ ) cells. For cell lines intended to contain G protein mutations, appropriate mutations can be artificially created / configured. (See US Pat. Nos. 6,168,927B1, 5,739,029 and 5,482,835, yeast cell lines).

  As a related aspect, the cells are transfected with a vector operably linked to a cDNA containing a sequence encoding the protein as described in SEQ ID NO: 2. Primary and secondary vectors containing such systems include 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, Calif.) And pPicZ (Invitrogen Corp, San Diego, Calif.).

  As a related aspect, host cells are transfected by such appropriate methods, and functional GAVE19 protein is expressed as a result of the transfection (see, eg, Sambrook et al., 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 form a complex with the G protein after expression, where the G protein controls second messenger formation. Not. Other methods for transfecting host cells utilized herein include transfection, electroporation, microinjection, microinduction, transfusion, cell fusion, DEAE. Dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of gene guns, or DNA vector transporters (eg, Wu et al., 1992, J. Biol. Chem. 267: 963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990), but is not limited thereto.

In the present invention, there are various uses of promoters. Indeed, the expression of a polypeptide according to the invention can be controlled by any promoter / enhancer element known in the state of the art, however these regulators must be functional in the host selected for expression. . Promoters used to regulate GAVE19 expression include the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the long repeat sequence at the 3 'end of Rous sarcoma virus. (Yamamoto et al., 1980, Cell 22: 787-797), herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natle. Acad. Sci. USA 78: 1441-1445), regulatory sequences of metallothionein genes ( Brinster et al., 1982, Nature 296: 39-42), prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727 -3731) or tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25), but is not limited thereto. The following documents can also be referred to. "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; promoter elements derived from yeast or other fungi such as the Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline Phosphatase promoter; animal transcriptional regulatory region with tissue specificity and used in transgenic animals: Elastase I gene regulatory region active in pancreatic endoplasmic reticulum cells (Swift et al., 1984, Cell 38: 639-646; Ormitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); Insulin gene regulatory region active in pancreatic β 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 regulatory region active in testis, breast, lymphocytes and mast cells (Leder et al., 1986, Cell 45: 485). 495), albumin gene regulatory region active in liver (Pinkert et al., 1987, Genes and Devel. 1: 268-276), α-fetoprotein gene regulatory region active in liver (Krumlauf et al., 1985, Mol) Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58), α1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devil. 1 : 161-171), β-globin gene regulatory region active in bone marrow cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94), poor brain Myelin basic protein gene regulatory region active in oligodendrocytes (Readhead et al., 1987, Cell 48: 703-712), myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 19 85, Nature 314: 283-286), and the gonadotropic gonadotropin releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

  Expression vectors containing nucleic acid molecules according to the present invention can be identified by four general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c). Presence or absence of selectable marker gene function, (d) expression of the inserted sequence. In the first approach, nucleic acids can be amplified by PCR to provide for detection of amplification products. In the second approach, the presence of a foreign 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 approach, the recombinant vector / host system is responsible for certain “selectable marker” gene functions (eg, β-galactosidase activity, thymidine kinase activity, antibiotic resistance, transduction) generated by inserting a foreign gene into the vector. Formation phenotype, presence of occlusion bodies in baculovirus, etc.) or identification and selection. In another embodiment, if a nucleic acid encoding a GAVE19 protein, variant thereof, or analog or derivative thereof is inserted into the “selectable marker” gene sequence of the vector, the set comprising the insert Recombinants can be identified by a defect in GAVE19 gene function. In the fourth approach, recombinant expression vectors can be used to determine the activity, biochemical or immunological properties of the gene product expressed by the recombinant, provided that the expressed protein is in a functionally active conformation. Can be identified by testing.

Various host / expression vector combinations may be employed for expression of the DNA sequences of the present invention. For example, useful expression vectors are composed of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40, and bacterial plasmids such as E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67: 31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, for example various derivatives of lambda phage, for example NM989, and other phage DNA, for example M13, and single-stranded filamentous phage DNA; Yeast plasmids such as 2μ plasmids or derivatives thereof; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; combinations of plasmids and phage DNA such as plasmids modified for combination with phage DNA Vector derived from the above, or other expression regulatory sequences.

  For example, in the baculovirus expression system, non-fusion transfer vectors include pVL941 (BamHI cloning site; Summers), pVL1393 (BamHI, SmaI, XbaI, EcoRI, NotI, XmaIII, BglII, and PstI cloning sites; Invitrogen), pVL1392. (BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI, and BamHI cloning sites; Summers and Invitrogen), and pBlueBacIII (BamHI, BglII, PstI, NcoI, and HindIII cloning sites, blue / white recombination available; Invitrogen ), And fusion transvectors include pAc700 (BamHI and KpnI cloning sites, where the BamHI recognition site begins with the start codon; Summers), pAc701 and pAc702 (same as pAc700 except for open reading frame), pAc360 (polyhedrin start codon) 36 base pairs B downstream of amHI cloning site; Invitrogen (195)), and pBlueBacHisA, B, C (3 different open reading frames, BamHI, BglII, PstI, NcoI, and HindIII cloning sites, N-terminal peptide for ProBond purification, and plaque blue / White recombination screening; Invitrogen (220)) can be used, but is not limited to these.

Mammalian expression vectors that can be used in the present invention include inducible promoters such as sihydrofolate reductase (DHFR), such as pED (PstI, SalI, SbaI, SmaI, and vectors that express both cloned genes and DHFR, and DHFR expression vectors such as EcoRI cloning sites; Kaufman, Current Protocols in Molecular Biology, 16.12 (1991)), or any expression vector of a DHFR / methotrexate coupled amplification vector. Alternatively, pEE14 (HindIII, XbaI, SmaI, SbaI, EcoI,
And at the BclI cloning site, where the vector expresses a glutamine synthase and the cloned gene; a glutamine synthetase / methionine sulphoximine coupled amplification vector such as Celltech).
In other embodiments, vectors that govern episomal expression under the control of Epstein-Barr virus (EBV), such as pREP4 (BamHI, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning sites, constructs RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamHI, SfiI, XhoI, NotI)
, NheI, HindIII, NheI, PvuII, and KpnI cloning sites, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen, pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamHI cloning site, induction Metallothionein Iia gene promoter, hygromycin selectable marker; Invitrogen), pREP8 (BamHI,
XhoI, NotI, HindIII, NheI, and KpnI cloning sites, RSV-LTR promoter,
Histidinol selection marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHI cloning sites, RSV-LTR promoter, G418 selection marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selection marker) N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen).
Selective mammalian expression vectors used in the present invention include pRc / CMV (HindIII,
BstXI, NotI, SbaI, and ApaI cloning sites, G418 selection; Invitrogen), pRc / RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selection; Invitrogen), and others. Vaccinia virus mammalian expression vectors used according to the present invention (see Kaufman, 1991 above) include pSC11 (SmaI cloning site, TK- and β-gel selection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI, SacII, KpnI, and HindIII cloning sites; TK- and β-gel selection) and pTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamHI, and Hpa cloning sites, TK or XPRT selection) Including, but not limited to.

Yeast expression systems can also be used to express the GAVE19 protein, variants thereof, or analogs or derivatives thereof, in accordance with the present invention. For example, unfused pYE32 vectors (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamHI, SacI, KpnI and HindIII cloning sites; Invitrogen) or fused pYESHisA, B, C (XbaI, SphI, ShoI,
Two of the following may be mentioned: NotI, BstXI, EcoRI, BamHI, SacI, KpnI, and HindIII cloning sites, N-terminal peptides purified with ProBond resin and cleaved with enterokinase; can do.

  Once a recombinant DNA molecule has been identified and isolated, several methods known in the art can be used for propagation. Once a suitable host system and growth conditions are established, the recombinant expression vector can be propagated and prepared quantitatively. As explained above, expression vectors that can be used include, but are not limited to, the following vectors or derivatives thereof: human or animal viruses such as vaccinia virus or adenovirus; Insect viruses such as; yeast vectors; bacteriophage vectors (eg, lambda), and plasmid and cosmid DNA vectors.

  In addition, host cell lines can be chosen to modulate the expression of the inserted sequences, or to modify and process the gene product in the specific fashion desired. Different host cells have specific and specific mechanisms for protein translation and post-translational processes and modifications (eg, glycosylation, cleavage (eg, of signal sequences)). Appropriate cell lines or host systems can be chosen to ensure the desired modification and process of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product.

Transgenic animals The host cells according to the invention can also be used to produce transgenic animals. For example, in certain embodiments, a host cell of the invention is a fertilized oocyte or embryonic stem cell into which a GAVE19 coding sequence has been introduced, such host cell having an exogenous GAVE19 sequence introduced into the genome. Can be used to create transgenic non-human animals, or homologous recombinant animals in which the endogenous GAVE19 sequence is mutated. Such animals are useful for studying the function and / or activity of GAVE19 and for identifying and / or evaluating modulators of GAVE19 activity. As used herein, “transgenic animal” refers to a non-human animal, preferably a mammal, in which one or more animal cells contain a transgene. Examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, rats, amphibians and the like.

The term “transgene” as used herein refers to exogenous DNA that has been incorporated into the genome of a cell in which the transgenic animal has grown and is maintained in the genome. The transgene governs the expression of the encoded gene product in one or more cell types or tissues of the transgenic animal.
As used herein, “homologous recombinant animals” refers to animals other than humans, preferably mammals, in which the endogenous GAVE19 gene is mutated by homologous recombination. It is then performed between the endogenous gene and the exogenous DNA molecule introduced into the animal cell (eg, the embryonic cell of the animal before it differentiates into an adult animal).

A transgenic animal according to the present invention can be created by introducing a nucleic acid molecule encoding GAVE19 into the male pronucleus of a fertilized oocyte using one of the transfection methods described above. Oocytes can then grow in foster mothers of pseudopregnant females. For example, a GAVE19 cDNA sequence, such as that of SEQ ID NO: 1, can be introduced as a transgene into the genome of a non-human animal.
Intron sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of transgene expression. Tissue specific regulatory sequences can be operably linked to the GAVE19 transgene to control GAVE19 protein expression in special cells. Methods for generating transgenic animals by embryo manipulation and microinjection (microinjection) are common knowledge, for example, other similar methods described in US Patent Nos. 4,736,866 and 4,870,009 and 4,873,191 can Expression of the transgene and / or GAVE19 mRNA in the genome in other tissues or cells can be used to create other transgenic animals. The first transgenic animal can then be used to breed another animal carrying the transgene. In addition, transgenic animals carrying a transgene encoding GAVE19 can also be bred to create other transgenic animals carrying other transgenes.

  In order to create a homologous recombination animal, the vector is prepared so as to contain at least a part of the GAVE19 gene into which a deletion, addition or substitution has been introduced, for example, in order to functionally mutate the GAVE19 gene. In a special embodiment, the vector is designed on homologous recombination such that the endogenous GAVE19 gene is functionally disrupted (ie can no longer encode a functional protein; also referred to as a “knockout” vector). You can also.

  Alternatively, the vector can be designed in homologous recombinants such that the endogenous GAVE19 gene is mutated or otherwise mutated but still encodes a functional protein (eg, The upstream regulatory region is mutated, resulting in a mutated expression of the endogenous GAVE19 protein).

  In the homologous recombination vector, the mutated portion of the GAVE19 gene is framed at the 5 ′ and 3 ′ ends by the additional nucleic acid sequence of the GAVE19 gene, and the exogenous GAVE19 gene carried by the vector and the embryonic stem cell Allows homologous recombination to occur between the endogenous GAVE19 genes. This additional GAVE19 flanking nucleic acid sequence is long enough for good homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5 'and 3' ends) are included in the vector (see, eg, Thomas et al., Cell (1987 for a description of homologous recombination vectors). ) 51: 503).

  The vector is introduced into an embryonic stem cell line (eg, by electroporation), and cells in which the introduced GAVE19 gene is homologously recombined with the endogenous GAVE19 gene are selected (eg, Li et al., Cell (1992) 69). : 915). When selected cells are injected into blastocysts of animals, aggregate chimeras are formed (see, for example, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL, Oxford, (1987) pp. 113-152). The chimeric embryo is then transferred to an appropriate pseudopregnant female adoptive parent and the embryo is brought to production. Offspring that have recombination DNA in embryonic cells can be used for animal breeding, and all cells of that animal contain DNA that has been homologously recombined by germline penetration of the transgene .

  For methods for producing homologous recombination vectors and homologous recombination animals, see Bradley, Current Opinion in Bio / Technology (1991) 2: 823-829 and PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968 and WO 93/04169.

  In another alternative embodiment, the non-human transgenic animal can be made to include a selective system that can control the expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of this cre / loxP recombinase system, reference can be made, for example, to Lakso et al., Proc Natl Acad Sci USA (1992) 89: 6232-6236. Another example of a recombinase system is the S. cerevisie FLP recombinase system (O'Gorrnan et al., Science (1991) 251: 1351-1355). If the cre / loxP recombinase system is used for transgene expression control, an animal containing a transgene encoding both the cre recombinase and the selected protein is required. Such animals can be supplied by the creation of “double” transgenic animals, for example one containing a transgene encoding the selected protein and the other containing a transgene encoding a recombinase 2 It can be made by crossing one transgenic animal.

The clones of non-human transgenic animals described here can also be obtained according to the methods described in Wilmut et al., Nature (1997) 385: 810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669. Can be created. Briefly, for example, somatic cells are isolated from a transgenic animal and induced to escape the growth cycle and enter the _Go phase. The stationary phase cells can then be fused to enucleated oocytes from the same species of animal from which the stationary phase cells were isolated using, for example, an electrical pulse. Then, the reconstructed oocyte is cultured, grown to morula or blastocyst, and then transplanted to a foster mother of pseudopregnant female. A child born from the female parent is, for example, an isolated somatic cell-derived clone.

Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, antibodies and fragments thereof according to the present invention can be used in one or more of the following methods: a) screening assays; b) detection assays. Methods (eg, chromosomal mapping, tissue classification, forensic biology); c) predictive medicine (eg, diagnostic assays, prognostic assays, clinical trial monitoring and pharmacogenomics): and d) therapeutic methods (eg, treatment and prevention). The GAVE19 protein interacts with other intracellular proteins and can therefore be used to (i) control cell proliferation; (ii) control cell differentiation; and (iii) control cell survival.
An isolated nucleic acid molecule according to the present invention can be used for GAVE19 protein expression (eg, via a recombinant expression vector in a host cell for gene therapy applications).
It can be used for detection of RNA (eg in biological preparations) or detection of genetic damage in the GAVE19 gene and for modulation of GAVE19 activity. In addition, GAVE19 protein can also be used to screen for agents or compounds that modulate GAVE19 activity or expression. Such drugs or compounds can be readily used for the treatment of inflammatory diseases such as rheumatoid arthritis, COPD (chronic obstructive pulmonary disease) and the like. Screening for GAVE19 protein production that is reduced or exhibits abnormal activity compared to the GAVE19 wild type protein can also be performed using the present invention. In addition, the anti-GAVE19 antibody according to the present invention can be used for detection and isolation of GAVE19 protein and also for modulation of GAVE19 activity. The present invention further extends to the novel agents identified by the above screening assays and their use as treatments as described herein.

Screening assay Activation of the G protein in the presence of endogenous ligands forms a G protein receptor complex and leads to GTP binding to the G protein. The GTPase domain of the G protein gradually hydrolyzes GTP to GDP, and under normal conditions the receptor is inactivated. However, constitutively activated receptors continue to hydrolyze GTP to GDP.

A substrate that is not hydrolyzed by G proteins, [ ³⁵ S] GTPγS, can be used to monitor enhanced binding to membranes expressing constitutively activated receptors. Traynor and Nahorski report that [ ³⁵ S] GTPγS can be used to monitor G protein binding to membranes in the presence and absence of ligand (Traynor et al., Mol Pharmacol ( 1995) 47 (4): 848-54). A preferred use of such an assay system is the primary screening of candidate compounds because it does not take into account the specific G protein that binds to the receptor for all G protein coupled receptors. This is because the system is generally available.

G _s20 stimulates the enzyme adenylyl cyclase, while G _i and _Go inhibit the enzyme.
It is well known in the art that adenylyl cyclase catalyzes the conversion of ATP to cAMP; therefore, constitutively activated GPCRs coupled to G _s proteins have been found to have increased cAMP cells. It is related to the inner level. Alternatively, constitutively activated GPCRs that may couple to G _i (or _Go ) proteins are associated with reduced intracellular levels of cAMP. "Indirect Mechanism of Synaptic Transmission", Chpt. 8, from Neuron to Brain (3 rd Ed.), Nichols et al. Sinauer Associates, Inc., see 1992. Therefore, assays that detect cAMP can be used to determine whether a candidate compound is an inverse agonist for the receptor. Many approaches known in the art can be used to measure cAMP. In some embodiments, anti-cAMP antibodies are used in an ELISA-basic format. In other embodiments, whole cell second messenger reporter system assays are contemplated (see PCT Publication No. WO 00/22131).

In a related aspect, cyclic AMP stimulates a cAMP-responsive DNA binding protein or transcription factor (CREB) and binds to a promoter at a specific site called a cAMP-responsive factor to direct the direction of gene expression. Promote gene expression. In this way, the reporter system can be configured such that a promoter containing multiple cAMP response elements is in front of a reporter gene such as, for example, β-galactosidase or luciferase. It should be noted that as the constitutively activated G _s -coupled receptor causes cAMP accumulation, it activates the expression of genes and reporter proteins. Reporter proteins such as β-galactosidase or luciferase are then detected by standard biochemical assays (PCT Publication No. WO 00/22131).

Other G proteins such as G _o and G _q is related to the activation of the enzyme phospholipase C, then the phospholipid is hydrolyzed to PIP2, 2 kinds of intracellular messengers: diacylglycerol (DAG) and inositol 1 , 4,5-triphosphate (IP3) is released. IP3 accumulation increase of G _q - associated receptors and G _o - is related to the activation of associated receptors (PCT Publication No. WO 00/22131). An assay to detect IP3 accumulation is that the candidate compound is G _q −
It can be used to determine whether it is an inverse agonist for the associated receptor or _Go -associated receptor. G _q -associated receptors can also be determined using an AP1 reporter assay that measures whether G _q- dependent phospholipase C causes activation of a gene containing the AP1 factor. In this way, G _q activated - associated receptor inverse agonist to reduce the expression, increasing the expression of the gene is demonstrated.

  Identify candidate compounds, test compounds or agents (eg, peptides, peptidomimetics, small molecules, or other agents) that bind to a modulator or GAVE19 protein or that promote or inhibit, for example, GAVE19 expression or GAVE19 activity A method (herein referred to as “screening assay”) is also provided.

  In certain embodiments, the invention provides methods for assaying candidate or test compounds that bind to or modify the membrane-bound form of GAVE19 protein, polypeptide, or biologically active portion thereof. . The test compounds of the present invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, but the library can be a biological library; Parallel library (solid phase or liquid phase); synthetic library methods that require deconvolution; “one-bead, single compound” library method; synthetic library methods using affinity chromatography selection. Biological library approaches are limited to peptide libraries, but the other four approaches are based on small molecule libraries of peptides, non-peptide oligomers or compounds (Lam, Anticancer Drug Des (1997) 12: 145). It can also be used.

  Examples of methods for synthesizing a molecular library include, 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. 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.

  The library of compounds can be a solution (eg, Houghten Bio / Techniques (1992) 13: 412-421) or beads (Lam, Nature (1991) 354: 82-84), Chips (Fodor, Nature (1993) 364: 555 ~ 556), bacteria (US Patent No. 5,223,409), spores (US Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA (1992) 89: 1865-1869) or phage (Scott et al., Science (1990) 249: 386-390; Devlin, Science (1990) 249: 404-406; Cwirla et al., Proc Natl Acad Sci USA (1990) 87: 6378-6382; and Felici, J Mol Biol (1991) 222: 301-310).

In a particular embodiment of the invention, the assay is a cell-based assay in which cells expressing GAVE19 protein, or a membrane-bound form of a biologically active portion thereof, on the cell surface are present. The ability to contact the test compound and bind the test compound to the GAVE19 protein is measured. The cell may be, for example, a yeast cell or a cell of mammalian origin. Measurement of the ability of a test compound to bind to the GAVE19 protein can be performed, for example, by conjugation with a test compound having a radioisotope or enzyme label, such as the GAVE19 protein of the test compound, or a biologically active portion thereof. The binding of can be measured by detecting the labeled compound in the complex. For example, the test compound can be labeled directly or indirectly with ¹²⁵ I, ³⁵ S, ¹⁴ C or ³ H, and the radioisotope can be detected by direct measurement of radioactive release or by scintillation counting. . Alternatively, the test compound can be enzymatically labeled, such as with horseradish peroxidase, alkaline phosphatase or luciferase, and the enzyme label can be detected by measuring the conversion of the appropriate substrate to product. it can.
In a specific embodiment, the assay involves contacting a known compound that binds to GAVE19 to form an assay mixture on the cell surface expressing the membrane-bound form of the GAVE19 protein or biologically active portion thereof. Contacting the test mixture with a test compound, and measuring the ability of the test compound to interact with the GAVE19 protein, wherein the measurement of the ability of the test compound to interact with the GAVE19 protein is determined using GAVE19 or This can be done by comparing the ability of the test compound to bind to its biologically active moiety with that of a known compound.

  In another embodiment, the assay is a cell-based assay on the surface of a cell expressing the membrane-bound form of the GAVE19 protein or biologically active portion thereof. Contacting the test compound and measuring the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of the GAVE19 protein or biologically active portion thereof. Measuring the ability of a test compound to modulate the activity of GAVE19 or a biologically active portion thereof can be done, for example, by measuring the ability to bind to a GAVE19 protein or interact with a GAVE19 target molecule. As used herein, “target molecule” refers to a molecule that GAVE19 protein actually binds to or interacts with, for example, a molecule on the surface of a cell expressing GAVE19 protein, or on the surface of a secondary cell. A molecule, a molecule in an extracellular fluid, a molecule associated with the inner surface of a cell membrane, or a cytoplasmic molecule. The GAVE19 target molecule may be a non-GAVE19 molecule or may be a GAVE19 protein or polypeptide according to the present invention. In some embodiments, the GAVE19 target molecule comprises a signaling pathway that facilitates transmission of extracellular signals (eg, signals generated by binding compounds to membrane-bound GAVE19 molecules) through the cell membrane and into the cell. It is a thing. The target may be, for example, a secondary intracellular protein having catalytic activity or a protein that facilitates association with downstream signal molecules with GAVE19.

Measuring the ability of a GAVE19 protein to bind to or interact with a GAVE19 target molecule can be performed using one of the methods described above that measure direct binding. In a particular embodiment, the ability of a GAVE19 protein to bind or interact with a GAVE19 target molecule can be determined by measuring the activity of the target molecule. For example, the activity of the target molecule can be determined by measuring the induction of target intracellular second messengers (eg, intracellular Ca ²⁺ , diacylglycerol, IP3, etc.), measuring the catalytic / enzymatic activity of the target against the appropriate substrate, reporter gene Measured by measuring the induction of (eg, a GAVE19 response regulator operably linked to a nucleic acid encoding a detectable marker, eg, luciferase), or by detecting an intracellular response, eg, cell differentiation or proliferation. be able to.

The present invention further includes a cell-free assay comprising contacting a test compound with a GAVE19 protein or biologically active portion thereof, and measuring the binding ability of the test compound to the GAVE19 protein or biologically active portion thereof. It extends to. GAVE19
The binding between the protein and the test compound can be measured directly or indirectly as described above. In a preferred embodiment, the assay method comprises contacting a GAVE19 protein or biologically active portion thereof with a known compound that binds to GAVE19 to form an assay mixture, and contacting the assay mixture with the test compound. And measuring the ability of the test compound to interact with the GAVE19 protein, wherein the ability of the test compound to bind to GAVE19 or a biologically active portion thereof is compared to that of a known compound It can be carried out.

  Other cell-free assays according to the invention involve contacting a test compound with a GAVE19 protein or biologically active portion thereof and modulating the activity of the GAVE19 protein or biologically active portion thereof ( For example, measuring the ability of a test compound to stimulate or inhibit). The ability of a test compound to modulate the activity of GAVE19 can be measured, for example, using one of the above-described methods for measuring the binding ability of GAVE19 protein to a GAVE19 target molecule. In other embodiments, the ability of a test compound to modulate the activity of GAVE19 can be determined by measuring the ability of the GAVE19 protein to further modulate the GAVE19 target molecule. For example, the catalytic / enzymatic activity of the target molecule when an appropriate substrate is used can be measured as described above.

  In addition, other cell-free assays according to the present invention include contacting a known compound that binds GAVE19 to GAVE19 protein or a biologically active portion thereof to form an assay mixture, and contacting the assay mixture with the test compound. And measuring the ability of the test compound to interact with the GAVE19 protein. Measuring the ability of the test compound to interact with the GAVE19 protein includes measuring the ability of the GAVE19 protein to bind or modulate suitably with the GAVE19 target molecule.

Receptors do not necessarily inhibit ligand binding, but by non-ligand molecules that cause structural changes in the receptor that allow G protein binding and possibly receptor aggregation, dimerization, or clustering that can lead to activation. Activated. For example, antibodies can be raised in various parts of GAVE19 exposed to the cell surface. These antibodies can activate cells via a cascade of G proteins as determined by standard assays such as monitoring cAMP levels or intracellular Ca ²⁺ levels. Monoclonal antibodies are more preferred because they involve molecular mapping and especially epitope mapping. Monoclonal antibodies are directed against both intact receptors expressed on the cell surface and peptides known to form on the cell surface. The method of Geysen et al., US Pat. No. 5,998,577 can be used to collect the majority of related peptides. Antibodies known to activate GAVE19 can be modified to minimize activities that are heterogeneous for GAVE19 activation, such as complement binding reactions. Thus, antibody molecules can be truncated or mutated to minimize or eliminate activities other than GAVE19 activation. For example, certain antibodies require only an antigen binding portion. In such cases, the _Fc portion of the antibody can be removed.

  Cells expressing GAVE19 become activated by exposure to antibodies. The activated cells can then be exposed to various molecules to identify which molecules modulate receptor activity, resulting in high or low activation levels. Molecules that achieve these goals can then be tested on cells expressing GAVE19 without antibodies to observe effects on non-activated cells. The target molecule is then tested and modified as a therapeutic candidate for a disorder associated with altered GPCR metabolism using known techniques.

The cell-free assay according to the present invention can be used as both a soluble form and a membrane-bound form of GAVE19. In the case of a cell-free assay comprising a membrane-bound type of GAVE19, it is desirable to use a solubilizing reagent in order to keep the membrane-bound type of GAVE19 in the solution. Such solubilizing reagents include n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methyl glucamide, decanoyl-N-methyl glucamide, TRITON X-100, TRITON X-114. , THESIT, isotridecyl poly (ethylene glycol ether) _n , 3-[(3-colamidopropyl) dimethylamino] -1-propanesulfonate (CHAPS), or N-dodecyl = N, N-dimethyl-3-ammonio Non-ionic surfactants such as -1-propanesulfonate are included.

In one or more embodiments of the above assay according to the present invention, separating the complex from one or both uncomplexes of the protein by immobilizing either GAVE19 or its target molecule. It is desirable to make it easy to cope with the automation of the test. Binding of the test compound to GAVE19 or the interaction of GAVE19 with the target molecule in the presence or absence of the candidate compound can be performed in any container suitable for containing the reaction mixture. Such containers include microtiter plates, test tubes, and microcentrifuge tubes. In some embodiments, a fusion protein can be provided to add a domain to which one or both proteins bind to the matrix. For example, glutathione-S-transferase / GAVE19 fusion protein or glutathione-S-transferase / target fusion protein can be adsorbed to glutathione SEPHAROSE beads (Sigma Chemical, St. Louis, MO).
Alternatively, glutathione-derivatized microtiter plates can be conjugated with test compounds. Thereafter, either the non-adsorbing labeled protein or the GAVE19 protein and the mixture are incubated under conditions that promote complex formation (eg, physiological conditions of salt and pH). After incubation, the beads or microtiter plate holes are washed to remove any unbound components and the complex formed is measured directly or indirectly. Alternatively, the complex can be dissociated from the matrix and the level of GAVE19 binding or activity measured using standard techniques.

  Other techniques for protein immobilization on a matrix can also be used in screening assays according to the present invention. For example, GAVE19 or any of its target molecules can be immobilized using conjugation of biotin and streptavidin. Biotin-conjugated GAVE19 or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, IL) And it can fix to each hole of 96-well plate (Pierce Chemicals) coated with streptavidin. Alternatively, antibodies that react with GAVE19 or a target molecule but do not interfere with binding of the GAVE19 protein to the target molecule can be derivatized to each well of the plate. During incubation, unbound target molecule or GAVE19 is captured in each well by antibody conjugation. Such complex detection methods include, in addition to those described above for GST-fixed complexes, immunodetection methods for complexes using GAVE19 or target molecules and active antibodies, and GAVE19 or target molecules There are solid-phase enzyme assays that rely on the detection of associated enzyme activity.

In other embodiments, modulators of GAVE19 expression can be identified by detecting GAVE19 mRNA or protein in cells contacted with the candidate compound. The expression or protein level of GAVE19 mRNA in the presence of the candidate compound is compared to the expression or protein level of GAVE19 mRNA in the absence of the candidate compound. Candidate compounds can be identified as modulators of GAVE19 expression based on the comparison. For example, if the GAVE19 mRNA expression or protein is greater in the presence (statistically significantly greater) than in the absence of the candidate compound, the candidate compound is an activator or agonist of GAVE19 mRNA or protein expression Is identified. Alternatively, if the expression of GAVE19 mRNA or protein is less present (statistically less significant) than in the absence of the candidate compound, the candidate compound inhibits GAVE19 mRNA or protein expression. Identified as a substance or antagonist. If GAVE19 activity decreases in the presence of a ligand or agonist, or constitutive GAVE19 is below baseline, the candidate compound is identified as an inverse agonist. The level of GAVE19 mRNA or protein expression in the cells can be detected by the methods described herein for GAVE19 mRNA or protein detection.

  In other aspects of the invention, the GAVE19 protein is a two-hybrid assay or a three-hybrid assay (eg, US Patent No. 5,283,317; Zervos 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 PCT Publication No. WO 94/10300) to identify other proteins that bind or interact with GAVE19 (“GAVE19-binding protein” or “GAVE19-bp”) and modulate GAVE19 activity. Can be used. Such GAVE19-binding proteins may also be involved in signal propagation by GAVE19 proteins, such as factors upstream or downstream of the GAVE19 pathway.

  Since the present invention enables mass production of pure GAVE19, it is possible to confirm the physical properties of the conformation of the region that seems to exert its function by rational drug design (pharmaceutical design). For example, the IC3 region and EC domain of a molecule are regions of particular interest. Once the shape and ionic configuration of the regions are identified, candidate compounds that interact with these regions are shaped and tested in complete cells, animals or patients. Methods that allow such 3-D structural information to be derived include X-ray crystal diffraction, NMR spectroscopy (nuclear magnetic resonance analysis), molecular modeling (molecular design), and the like. 3-D structures can also lead to the identification of similar conformational sites in other known proteins where there are sites where known drugs act. These drugs or their derivatives would find use in the treatment of inflammatory diseases or disorders such as rheumatoid arthritis or COPD (chronic obstructive pulmonary disease).

  The invention further extends to the novel agents identified by the screening assays described above and their use for treatment as described herein.

Test method according to the present invention Detection Assay Methods DNA sequence portions or fragments according to the present invention can be used in various ways as polynucleotide reagents. For example, the sequence may include (i) mapping of each gene on a chromosome and searching for a genetic region associated with a genetic disease; (ii) identifying an individual from a small biological specimen (tissue typing); It can be used as an aid for forensic identification of preparations. These uses are detailed below.

1. Chromosome mapping Once the sequence of a gene (or part of a sequence) has been isolated, the sequence is
Can be used to map the location of the E19 gene. Therefore, the GAVE19 nucleic acid molecules described herein or fragments thereof can be used to map the location of GAVE19 in the genome. Mapping the arrangement of the GAVE19 sequence in the genome is an important step in showing the causal relationship between the disease and the gene-related disease.

  Briefly, the GAVE19 gene can be mapped into the genome by preparing PCR primers (preferably 15-25 bp in length) from the GAVE19 sequence. The primers are used for PCR screening of somatic cell hybrids containing individual mouse chromosomes. Then, a fragment in which only the hybrid containing the mouse gene corresponding to the GAVE19 sequence is amplified is prepared.

Special methods include, but are not limited to, denaturing mouse chromosomes and then contacting the detectably labeled GAVE19 DNA molecule under stringent hybridization conditions. Hybridization and sequencing of detectably labeled GAVE19 DNA molecules reveals the location of GAVE19 in the mouse chromosome. Such in situ hybridization techniques are described in Fan et al., Proc Natl Acad Sci USA (1990) 87: 6223-27, in which are labeled flow-sorted chromosomes. And preselection by hybridization with a chromosome-specific cDNA library. Can be used to provide a precise chromosomal arranged in in situ hybridization (FISH) is also a stage in DNA sequence of a cell metaphase to chromosomes dispersion. Chromosomal dispersions can be created using compounds that inhibit mitotic spindle formation, such as cells in which cell division is blocked in metaphase by colcemid. Chromosomes are briefly treated with trypsin and then stained with Giemsa. A light and dark band pattern develops on each chromosome, so that the chromosomes can be identified individually. The FISH method can also be performed on short DNA sequences of 500 to 600 bases or less. However, clones longer than 1,000 bases are highly likely to bind to unique chromosomal coordinates, and their signal strength is sufficient for simple detection methods. Preferably 1,000 bases and more preferably 2,000 bases are necessary to obtain good results in a reasonable time. For an overview of this technology, reference is made to Verma et al. (Human Chromo somes: A Manual of Basic Techni-ques (Pergamon Press, New York, 1988)). Chromosome mapping can be inferred in silico and can take advantage of statistical considerations such as rod scores or simply proximity.

  Chromosome mapping reagents can be used individually to locate a single site on a chromosome. Furthermore, a panel of reagents can be used to mark multiple sites and / or multiple chromosomes. The reagent corresponding to the region where the GAVE19 gene is arranged is actually suitable for the purpose of mapping. The coding sequence appears to be conserved within the gene family, thus increasing the chance of cross-hybridization during chromosomal mapping.

Once a sequence has been mapped to the correct chromosomal configuration, the physical configuration of the sequence on the chromosome is correlated with the genetic map data. The correlation between genes mapped to the same chromosomal region and disease can therefore be determined by the linkage analysis described for example in Egeland et al., Nature (1987) 325: 783-787 (of physically adjacent genes). (Co-inherited).

It is also possible to detect a difference in DNA sequence between an animal suffering from a disease involving GAVE19 and an animal not suffering from the disease. If a mutation is observed in some or all animals suffering from a disease, but not in an unaffected animal,
The mutation is thought to cause the particular disease. A comparison between diseased and unaffected animals is usually first looking for structural changes in the chromosome, which can be detected, for example, by PCR, which is visible from chromosomal variance or based on DNA sequence A defect or metastasis that can occur. Finally, to confirm that the presence of the mutation and the mutation can be distinguished from the polymorphism by performing the complete sequence of the genes from several animals.

1. Diagnostic Assays An exemplary method for detecting the presence or absence of GAVE19 in a biological preparation is to obtain the biological preparation from a test animal and to use a GAVE19 protein or a nucleic acid encoding a GAVE19 protein (eg, mRNA or genome). DNA), and as a result, contacting the biological preparation with a compound or reagent capable of detecting the presence of GAVE19 in the biological preparation. Preferred reagents for detecting GAVE19 mRNA or genomic DNA include labeled nucleic acid probes that can hybridize to GAVE19 mRNA or genomic DNA. The nucleic acid probe is an oligonucleotide having a full length GAVE19 nucleic acid or a portion thereof, such as the nucleic acid of SEQ ID NO: 1, for example at least 15, 30, 50, 100, 250 or 500 or more nucleotides in length. Specifically hybridizes to GAVE19 mRNA or genomic DNA under mild conditions.

The special reagent for detecting the GAVE19 protein is an antibody capable of binding to the GAVE19 protein, preferably a detectably labeled antibody. The antibody is polyclonal, chimeric or more preferably monoclonal. An intact antibody, or a fragment thereof (eg, F _ab or F _(ab ′ _{) 2} ) can be used.
The term “biological preparation” refers to tissues, cells and cell fluids isolated from a subject, but also includes tissues, cells and cell fluids present in a subject. That is, the detection method according to the present invention can be used to detect GAVE19 mRNA, protein or genomic DNA in a biological preparation not only in vivo but also in vitro . For example, in vitro methods for detecting GAVE19 mRNA include Northern hybridization and in situ hybridization.
In vitro methods for detecting GAVE19 genomic DNA include Southern hybridization. Furthermore, the in vivo method for detecting GAVE19 protein involves the introduction of labeled anti-GAVE19 antibody into the animal body. For example, the antibody is labeled with a radioactive marker, and its presence and location in the animal can be detected by standard imaging techniques.

  In other embodiments, the biological preparation contains protein molecules from a test animal. Alternatively, the biological preparation contains mRNA molecules from the test animal or genomic DNA molecules from the test animal. The special biological sample used here is a white blood sample of peripheral blood isolated from an animal body by a conventional method.

  In other embodiments, the method can further obtain a biological preparation from a control animal, detect GAVE19 protein, mRNA or genomic DNA, such that the presence and amount of GAVE19 protein, mRNA or genomic DNA is present. Contacting the biological preparation with a compound or reagent capable of detecting in the biological preparation, and the presence and amount of GAVE19 protein, mRNA or genomic DNA in the control preparation in the test preparation GAVE19 protein, mRNA Or consisting of determining whether the compound modulates the expression or activity of GAVE19 as compared to the presence and amount of genomic DNA.

Chemical Library High Throughput Assay Any assay for compounds capable of modulating the activity of GAVE19 can be applied to high productivity screening. High productivity screening systems are commercially available (eg, Zymark Corp., HopKinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc., Fullerton, CA; Precision Systems, Inc., Natick, MA). Etc.). These systems are typically automated throughout the process, including sample and reagent hippet collection, liquid dispensing, timed incubation, and final reading of the microplate in a detector compatible with the assay. These combinable systems allow high throughput and quick start of measurement, as well as high adaptability and custom manufacturing. Manufacturers of such systems offer various high productivity detailed protocols. For example, Zymark Corp. provides a technical report describing screening systems for detecting gene transcription, ligand binding, and other modulations.

Kit The present invention also extends to a kit for detecting the presence of GAVE19 in a biological preparation (test sample). Such kits can be used to determine whether a particular compound modulates GAVE19 expression or activity. For example, the kit includes a labeled compound or reagent capable of detecting GAVE19 protein or mRNA in a biological preparation, and a method for measuring the amount of GAVE19 in the preparation (eg, anti-GAVE19 antibody or GAVE19, eg And an oligonucleotide probe that binds to DNA encoding SEQ ID NO: 1.

  In the case of an antibody-based kit, the kit includes, for example, (1) a primary antibody that binds to the GAVE19 protein (eg, bound to a solid support); It consists of different antibodies that bind to a 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 other molecules that bind to the primary antibody can be detectably labeled. In any case, what is labeled and bound has a role as a detectable reporter molecule, as is known in the art.

  In the case of an oligonucleotide-based kit, the kit according to the present invention is, for example: (1) an oligonucleotide such as a detectably labeled oligonucleotide that hybridizes to the GAVE19 nucleic acid sequence, or (2) a GAVE19 nucleic acid molecule Can be composed of a pair of primers useful for amplifying. The kit can further include, for example, a buffer, a preservative, or a protein stabilizer. The kit can also include components necessary to detect a detectable reagent (eg, an enzyme or a substrate). Moreover, the kit can also include a control sample or a control sample series for testing and comparison with the test sample.

2. Pharmacogenomics As explained herein, GAVE19 expression is regulated in cells that are activated or involved in inflammatory conditions. Disorders associated with inflammation include anaphylactic conditions, colitis, Crohn's disease, edema conditions, contact hypersensitivity, allergies, other forms of arthritis, meningitis and other conditions where the immune system is swollen and Responds to seizures caused by vasodilation, heat, aggregated cells, fluids, and the like in similar conditions. Thus, reagents or modulators that have a stimulatory or inhibitory effect on GAVE19 activity (eg, GAVE19 gene expression) as identified by the screening assays described herein may cause disorders (eg, asthma, chronic obstruction). Can be administered individually as a treatment (prophylactic or therapeutic) for idiopathic lung disease or inflammation involving rheumatoid arthritis). In connection with such treatment, the pharmacogenomics of the individual (ie, study of the correlation between the individual's genomic type and the individual's response to the foreign compound or drug) is considered. Differences in the metabolism of the therapeutic agent can lead to severe side effects or treatment failure due to changes in the correlation between dosage and blood concentration of the pharmacologically active agent. Thus, the pharmacogenomics of an individual allows for the selection of effective reagents (eg, drugs) for prophylactic or therapeutic treatment taking into account the individual's genomic type. Such pharmacogenomics can also be used to determine appropriate dosages and therapies.

Pharmacogenomics also deals with clinically important genetic variations in drug response due to drug processing variations and abnormal effects in affected individuals. See, for example, Linder, Clin Chem (1997) 43 (2): 254-266. In general, two types of pharmacogenomic conditions can be distinguished. A genetic condition that is passaged as a single factor that varies the way a drug acts on the body is called a “drug-action mutation”. A genetic condition that is passaged as a single factor that varies the way the body acts on a drug is called a “drug metabolism mutation”. Pharmacogenomic conditions occur as either rare defects or polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a well-known genetic enzyme disease in which the major clinical complications are oxidants (antimalarial drugs, sulfa drugs, Erythrocyte hemolysis after ingestion of painkillers or nitrofurans and feeding of fava beans.

  As an illustrative state, the activity of drug metabolizing enzymes is a major determinant of both the intensity and time of drug action. The discovery of genetic polymorphisms of drug-metabolizing enzymes (eg, N-acetyltransferase 2 (NAT2) and cytochrome P450 enzymes, CYP2D6 and CYP2C19) is the reason why some patients expected to take standard and safe doses of drugs as expected It provides an explanation for the question of efficacy or excessive drug response and serious toxicity. A polymorphism is expressed in two phenotypes in an individual, a strong metabolite (EM) and a weak metabolite (PM). The prevalence of PM varies between different individuals. For example, the gene encoding CYP2D6 is highly polymorphic, and several mutations have been identified in PM, all of which are known to be lacking functional CYP2D6. The weak metabolites of CYP2D6 and CYP2C19 very often experience excessive drug response and side effects when standard doses are taken. If the metabolite is an active therapeutic agent, PM will not show a therapeutic response, which has been demonstrated for the analgesic effect of codeine mediated by the metabolite produced by CYP2D6, morphine. Another extreme example is the so-called super express metabolite, which does not respond at all to standard doses. Recently, it has been identified that the molecular basis of super express metabolism is due to CYP2D6 gene amplification.

  The activity of a homologue of the GAVE19 protein or the expression of a DNA molecule that is a homologue of a GAVE19 nucleic acid in an individual can be determined in order to select suitable reagents for the therapeutic or prophylactic treatment of the individual. In addition, pharmacogenomic research can also be used for genomic typing of polymorphic alleles encoding drug metabolizing enzymes to identify individual drug response phenotypes. it can. Findings when utilized in dosage determination or drug selection can avoid side effects or treatment failure and therefore of molecules identified using one of the exemplary screening assays described herein. When treating a subject with a GAVE19 molecule, the therapeutic or prophylactic effect can be increased.

D. Methods of Treatment As explained above, the present invention extends to assays for identifying agents or reagents that modulate the expression of GAVE19 in mice.
Aspects of GAVE19 expression, ie, high expression in the normal spleen, increased the level of expression in collagen-induced arthritis (CIA) RA mice spleen compared to normal spleens, and in the lungs of CIA RA mice Since the expression level increased by more than 5 times compared to normal lung, such drugs or reagents may cause diseases or disorders such as inflammatory disorders (eg asthma), chronic obstructive pulmonary disease and rheumatoid arthritis. It can be easily used for the treatment of this.

1. Prophylaxis In one aspect, the present invention provides a method for preventing a disease or symptom as described above by administering to a subject an agent that modulates one of GAVE19 expression or at least one GAVE19 activity. is there. Those who benefit from such treatment can be identified, for example, by any or combination of diagnosis or prognosis well known to those skilled in the art. In order to prevent or delay the prevention or progression of a disease or disorder, administration of a prophylactic agent can be performed before the symptoms of such disease or disorder become apparent.

2. The reagents used in the treatment of treatment inflammatory diseases, i.e. those capable of modulating the activity of GAVE19 protein in mice, nucleic acid or protein, of the same type of naturally occurring GAVE19 protein ligand, a peptide, GAVE19 peptide-like or other Like a small molecule, a reagent as described herein. In certain embodiments, the reagent stimulates one or more biological activities of the GAVE19 protein. Examples of such stimulants include GAVE19 protein incorporated into cells and nucleic acid molecules encoding GAVE19. In other embodiments, there are reagents that inhibit one or more biological activities of the GAVE19 protein. Examples of such inhibitors are antisense GAVE19 nucleic acid molecules and anti-GAVE19 antibodies. Alternative methods include in vitro (eg, by culturing cells with reagents) or in vivo ( in
vivo ) (for example, by administering a reagent to a subject). As such, the present invention provides reagents (eg, reagents identified by the screening assays described herein) or mixing of reagents that modulate GAVE19 expression or activity in mice (eg, upstream or downstream control). Provided is a method of treating an individual suffering from a disease or disorder as described above comprising administering an agent to the individual. Another aspect involves administering a GAVE19 protein or nucleic acid molecule as a treatment for the method.

  The present invention will be better understood by the exemplary examples of the invention given below, rather than as a limiting listing. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. However, it is not meant to limit the scope of the invention.

  Many G protein-coupled receptors (GPCRs) are targeted by about 50% of today's therapeutics on the market. GPCRs are activated by a wide range of ligands including peptides, neurotransmitters, hormones, growth factors, amines, lipids, fatty acids, odorants and light. Instability of GPCR action results in many pathological conditions. GAVE19, an ortholog of human GAVE18 that is highly expressed in the normal spleen, shows an expression level in the RA mouse spleen of collagen-induced arthritis (CIA) that is up to 2-fold increased compared to the normal spleen. Expression levels increased by more than 5 times compared to normal lung in CIA RA mouse lung. Thus, GAVE19 provides a novel pharmaceutical agent that targets drugs or reagents used in the treatment of diseases or disorders such as inflammatory disorders (eg, asthma), chronic obstructive pulmonary disease, and rheumatoid arthritis.

  The identification and cloning of GAVE19 also provides an opportunity to find endogenous ligands through a de-orphaning process. Natural or surrogate ligands identified by de-orphaning provide a tool for screening for molecules that activate or block receptor signaling and thus alter intracellular physiology or function.

Materials and methods
Identification and cloning of GAVE19 : The mouse homologous DNA sequence could not be revealed using the human GAVE18 DNA sequence superimposed on the mouse genomic DNA database. Therefore, human GAVE18 DNA containing the coding region was used as a probe for screening the mouse genome BAC library of Research Genetics. Multiple DNA primers designed according to the human GAVE18 sequence were used to sequence positive mouse BAC. Unique primer, 5'GGC TTC CCC CAA AGA C
AA AG3 '(SEQ ID NO: 3) provided mouse GAVE19 DNA sequence data. Thus, multiple mouse DNA sequence primers were designed for primer walking to determine the sequence of the mouse GAVE19 coding region.

Mouse disease model and RNA isolation : Isolation of mouse total RNA from various tissues and organs was performed using the Trizol reagent from GIBCO BLR, based on manufacturing instructions. Total RNA was converted to cDNA using ABI's Multiscribe RT-PCR kit.

TaqMan analysis : TaqMan reactions were performed in duplicate with mouse tissue cDNA and the GAPDH gene was used as an internal control. TaqMan results were then calculated as relative expression. Relative expression is (2 ^{^} (0-ddCt)) ^* 1000, where ddCT is (GAVE1
9 average value Cts-GAPDH average value Cts)-(GAVE19 average value NTC Cts-G
APDH average value NTC Cts). TaqMan probes were synthesized by Operon Technologies on a custom basis. TaqMan primer sequence 1 is: 5 ′ CTG TTC TTG CTG GTG AAA ATG AA 3 ′ (SEQ ID NO: 4) and sequence 2 is 5 ′ CCA T
GA ACC ACC ACG AGG TT3 ′ (SEQ ID NO: 5). The TaqMan probe sequence is 5 '(Fam) -TCA CGT TCA GTG ACC ACC ATG GCT
G- (Tet) 3 '(SEQ ID NO: 6). TaqMan reaction is 96-well plate MicroAmp
Performed in an optical tube (PE).

Results Description The TaqMan expression profile shows that the level of GAVE19 expression in the joints of the CIA (collagen-induced arthritis) RA model is gradually increasing from normal control samples to RA grade 4. The higher the RA grade in the joint, the higher the correlation of GAVE19 expression. GAVE19 has also been shown to significantly increase expression levels in the CIA RA lung and spleen compared to normal control lung and spleen. In a mouse EAE (experimental allergic encephalomyelitis) model of multiple sclerosis, GAVE19 has been shown to increase expression levels in the brain during the EAE peak period compared to expression levels in the brain during the preclinical stage .

  The expression profile data is also displayed as a graph in FIG. All these data indicate that GAVE19 plays an important role in inflammation-related diseases.

Discussion Human GAVE18 and a novel mouse G-coupled receptor GAVE19, orthologous, have been identified and cloned. The full length coding region of the DNA and the amino acid sequence deduced therefrom were identified. The tissue distribution of the receptor was analyzed by RT-PCR (TaqMan) analysis in normal control mice and collagen-induced arthritis (CIA) mice. The expression profile of GAVE19 showed similarity to GAVE18 (human ortholog) and its important role in inflammatory diseases like rheumatoid arthritis was pointed out. Therefore, by using the various assays described herein, GAVE19 provides a new pharmaceutical target for inflammatory diseases such as asthma, RA (rheumatoid arthritis), COPD (chronic obstructive pulmonary disease). It should be noted that the compounds and reagents that modulate the expression and / or activity of GAVE19 in mice found by the assay method according to the present invention are considered to be readily usable for the treatment of such diseases.

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

Furthermore, it should be understood that all base sizes or amino acid sizes, and all molecular weights or molecular mass values given for nucleic acids or polypeptides are approximate and presented for illustrative purposes.
Various documents are cited herein, which are disclosed as they are as cited documents.

A DNA sequence encoding GAVE19 (SEQ ID NO: 1). Amino acid sequence of GAVE19 (SEQ ID NO: 2). GAVE19 expression profile with MPD 1.1.2. GAVE19 expression profile with MPD 1.1.2. Comparison of amino acid sequences between GAVE19 (SEQ ID NO: 2) and human ortholog GAVE18 (SEQ ID NO: 7).

Claims (28)

  An isolated nucleic acid molecule comprising the DNA sequence of Figure 1 (SEQ ID NO: 1).   An isolated nucleic acid molecule capable of hybridizing to the isolated nucleic acid molecule of claim 1 under stringent hybridization conditions, or a hybridization probe complementary to the isolated nucleic acid molecule of claim 1.   The isolated nucleic acid molecule according to claim 1 or 2, which encodes a polypeptide having the amino acid sequence of Fig. 2 (SEQ ID NO: 2).   3. The isolated nucleic acid molecule of claim 2, which encodes a polypeptide having an amino acid sequence that exhibits at least 30% identity 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 detectably labeled isolated nucleic acid molecule of claim 5, wherein the detectable label comprises an enzyme, a radioisotope or a chemical that generates fluorescence.   A purified polypeptide comprising 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.   10. The purified polypeptide of claim 9, wherein the detectable label comprises an enzyme, a radioisotope, or a chemical that generates fluorescence.   An antibody having the purified polypeptide according to claim 7 as an immunogen.   The antibody according to 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, which is detectably labeled.   14. The antibody of claim 13, wherein the detectable label comprises an enzyme, a radioisotope or a chemical that produces fluorescence.   An expression vector operatively associated with an expression regulator and comprising the isolated nucleic acid molecule of claim 1.   An expression vector operatively associated with an expression regulator and comprising the isolated nucleic acid molecule of claim 2.   The expression vector according to claim 15 or 16, wherein the expression regulator 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 regulator is a promoter. Promoters are hCMV immediate 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 system, trp system, TAC system, TRC system, lambda phage major operator and promoter region, 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, comprising:   A host cell transformed or transfected with the expression vector of claim 15 or 16.   The host cell according to claim 20, wherein the host cell is a prokaryotic cell or a eukaryotic cell. Host cells are E. coli (E. Coli), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Streptomyces (Streptomyces), yeasts, CHO, R1.1, B-W , L-M, COS1, COS7, BSC1, BSC40 23. The host cell of claim 21, comprising a BMT10 or Sf9 cell. The following steps:
a) culturing the host cell of claim 20 under conditions such that the isolated polypeptide is expressed; and b) isolating the isolated polypeptide from the host, the culture, or a combination thereof. A process for producing a purified polypeptide according to claim 7 comprising recovering.   Whether a potential agonist is contacted with a cell expressing GAVE19, and whether the signaling activity of GAVE19 in the presence of the potential agonist is increased relative to the activity of GAVE19 in the absence of the potential agonist A method of identifying an agonist of GAVE19, comprising:   Contacting a potential inverse agonist with cells expressing GAVE19, and whether the activity of GAVE19 in the presence of the potential inverse agonist is reduced compared to the activity of GAVE19 in the absence of the potential inverse agonist And determining whether it decreases in the presence of an endogenous ligand or agonist, a method of identifying an inverse agonist of GAVE19.   Whether a potential antagonist is contacted with a cell expressing GAVE19, and whether the signaling activity of GAVE19 in the presence of the potential antagonist is reduced compared to the activity of GAVE19 in the presence of an endogenous ligand or agonist A method for identifying antagonists of GAVE19, comprising measuring whether or not.   A therapeutic composition comprising an agonist, antagonist, or inverse agonist of GAVE19 capable of modulating GAVE19 signaling activity or transmission.   A method of treating a disease comprising administering to a patient in need thereof a therapeutic composition comprising an agonist, antagonist or inverse agonist of GAVE19 capable of modulating GAVE19 signaling activity or transmission.

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