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WO2003008560A2 - Nouveau recepteur couple aux proteines g, gave8 - Google Patents

Nouveau recepteur couple aux proteines g, gave8 Download PDF

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Publication number
WO2003008560A2
WO2003008560A2 PCT/US2002/023208 US0223208W WO03008560A2 WO 2003008560 A2 WO2003008560 A2 WO 2003008560A2 US 0223208 W US0223208 W US 0223208W WO 03008560 A2 WO03008560 A2 WO 03008560A2
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Prior art keywords
gave8
protein
nucleic acid
cell
activity
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PCT/US2002/023208
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English (en)
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WO2003008560A8 (fr
Inventor
Haifeng Eishingdrelo
Jidong Cai
Ali Ardati
Anthony Sandrasagra
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Aventis Pharmaceuticals Inc,
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Publication date
Priority claimed from GBGB0125704.7A external-priority patent/GB0125704D0/en
Application filed by Aventis Pharmaceuticals Inc, filed Critical Aventis Pharmaceuticals Inc,
Priority to EP02747073A priority Critical patent/EP1448583A4/fr
Priority to CA002454427A priority patent/CA2454427A1/fr
Priority to MXPA04000437A priority patent/MXPA04000437A/es
Priority to BR0211338-4A priority patent/BR0211338A/pt
Priority to JP2003514877A priority patent/JP2005522184A/ja
Priority to IL15993902A priority patent/IL159939A0/xx
Priority to KR10-2004-7001067A priority patent/KR20040017831A/ko
Publication of WO2003008560A2 publication Critical patent/WO2003008560A2/fr
Priority to NO20040252A priority patent/NO20040252L/no
Publication of WO2003008560A8 publication Critical patent/WO2003008560A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/08—Bronchodilators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • GPCRs The G protein-coupled receptors
  • GPCRs are a large family of integral membrane proteins that are responsible for cellular signal transduction. GPCRs respond to a variety of extracellular signals, including neurotransmitters, hormones, odorants and light and are capable of transducing signals across the cell membrane so as to initiate a second messenger response within the cell.
  • GPCRs are characterized by an extracellular ligand binding domain, seven transmembrane domains and an intracellular domain that interacts with cellular elements associated with signaling.
  • the functions the receptors perform, such as binding ligands and G proteins, are associated to the presence of certain amino acids in critical positions.
  • Edg GPCRs show amino acid sequence identity of 31% to 34% as a subfamily, but contain two homology clusters with greater similarity of structure and function.
  • One homology cluster includes Edg-2, Edg-4, Edg-7 and Edg-8 proteins, that bind lysophosphatidic acid (LPA) but not sphingolipids.
  • a second homology cluster encompasses Edg-1, Edg-3 and Edg-5 that bind sphingosine-l -phosphate (SIP or SPP) but not LPA (Goetzl et al., Adv Exp Med Biol (1999) 469:259-264).
  • SIP is a potent, extracellular lysolipid phosphoric acid mediator that is released, for example, during platelet activation (Moser et al., J Cell Biol (1992) 116:1517-1526). SIP elicits a wide variety of responses by cells, prominent among those are cell proliferation (Zhang et al, J Cell Biol (1991) 114:155-167; Bornfeldt et al., J Cell Biol (1995) 130:193-206; and Berger et al., Mol Pharmacol (1996) 50:451-457) and anti-apoptosis (Cuvillier et al., Nature (1996) 381 :800-803; Edsall et al., J Neurosci (1997) 17:6952-6960).
  • GPCRs Given the role GPCRs have in disease, and the ability to treat disease by modulating the activity of GPCRs, identification and characterization of previously unknown GPCRs can provide new compositions and methods of treatment for disease states that involve the activity of a GPCR.
  • the instant invention identifies and characterizes the expression of a novel GPCR, GANE8, and provides compositions and methods for applying the discovery to the identification and treatment of related diseases.
  • the instant invention relates to a newly identified G protein-coupled receptor.
  • the instant G protein-coupled receptor is expressed specifically in spleen and brain. Further the expression in the brain is localized in the white matter. That and other information intimates that the novel receptor, identified as GAVE8, is involved in a variety of inflammatory diseases, including multiple sclerosis and various perturbations of the immune system, as well as in disorders of the nervous system, and particularly the central nervous system.
  • the invention relates to isolated nucleic acids selected from the group consisting of an isolated nucleic acid which encodes a vertebrate protein of amino acids as set forth in SEQ ID NO:2, variants, mutations and fragments thereof, and an isolated nucleic acid which comprises a nucleotide sequence as set forth in SEQ ID NO:l, variants, mutations and fragments thereof. Further, the invention relates to nucleic acid hybridization probes and complementary fragments which bind to SEQ ID NO:l or hybridization probes and complementary fragments which bind to nucleic acids which encode the amino acid sequence as set forth in SEQ ID NO:2.
  • the invention relates to nucleic acids having about 90% -99% identity to SEQ ID NO:l, including nucleic acids having about 90% -99% identity to isolated nucleic acids encoding an amino acid sequence as set forth in SEQ ID NO:2.
  • the ohgonucleotides comprise at least 8 nucleotides and methods of hybridizing are contemplated comprising the steps of contacting the complementary oligonucleotide with a nucleic acid comprising the nucleotides as set forth in SEQ ID NO:l under conditions that permit hybridization of the complement with the nucleic acid.
  • complementary fragments may serve as anti-sense ohgonucleotides for methods of inhibiting the expression of GAVE8, in vivo and in vitro.
  • Such methods may comprise the steps of providing an oligonucleotide sequence consisting of the complement of the nucleotides as set forth in SEQ ID NO: 1, providing a human cell comprising an mRNA compromising the sequence of nucleotides as set forth in SEQ ID NO:l and introducing the oligonucleotide into the cell, where the expression of GAVE8 is inhibited by mechanisms which include inhibition of translation, triple helix formation and/or nuclease activation leading to degradation of mRNA in the cell.
  • the invention also relates to isolated polypeptides selected from the group consisting of purified polypeptides of amino acid sequence as set forth in SEQ ID NO:2, variants, mutations and fragments thereof, and purified polypeptides having additional amino acid residues which provide functional properties to the polypeptide.
  • the invention further relates to the nucleic acids operably linked to expression control elements, including vectors comprising the isolated nucleic acids.
  • the invention further relates to cultured cells transformed to comprise the nucleic acids of the invention and methods for producing a polypeptide comprising the steps of growing transformed cells comprising the nucleic acids of the invention, permitting expression and purifying the polypeptide from the cell or medium in which a cell was cultured.
  • a further aspect of the invention includes an isolated antibody that binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies. Further, in a related aspect, methods of producing antibodies and methods for treating GAVE8 related diseases with an antibody that binds to GAVE8 are disclosed.
  • An additional aspect of the invention includes methods, for diagnostic purposes, for determining the presence or absence of GAVE8 in a biological and/or tissue sample.
  • therapeutic methods for modulating GAVE8 signal transduction, including administration of peptides, agonists, antagonists, inverse agonists and/or antibody to a patient in need thereof.
  • methods for identifying modulators of GA VE8 comprising the steps of providing a chemical moiety, providing a cell expressing GAVE8 and determining whether the chemical moiety modulates the signaling activity of GANE8, including whether such modulation occurs in the presence or absence of an endogenous ligand.
  • the chemical moieties can include, but are not limited to, peptides, antibodies, agonists, inverse agonists and antagonists.
  • compositions include nucleic acids, antibodies, polypeptides, agonists, inverse agonists and antagonists.
  • methods of the invention also include methods of treating disease states and modulating GAVE8 signaling activity by administering such therapeutic compositions to a patient in need thereof.
  • the instant invention relates to a method of inferring and determining GAVE8 structure and function in neural cells by testing peripheral blood cells.
  • Figure 1 provides a nucleic acid sequence of GAVE8 (SEQ ID NOT).
  • Figure 2 depicts an amino acid sequence of GAVE8 (SEQ ID NO:2).
  • Figure 3 is a computerized representation of a Northern blot of RNA obtained from various human tissues.
  • Figure 4 is a computerized representation of a Northern blot for GAVE8 expression in various parts of the brain.
  • Figure 5a provides the GAVE8 TaqMan" expression profile in various regions of the brain.
  • Figure 5b provides the GAVE8 TaqMan ® expression profile in hypothalamus.
  • Figure 6a provides the GAVE8 TaqMan ® expression profile in peripheral tissues.
  • Figure 6b provides the GAVE8 Taqman ® expression profile in peripheral tissues.
  • Figure 7 depicts the change in luciferase readout in an assay using
  • GAVE8-expressing cells HEK transformants, a modified 293 cell line that expresses a
  • SIP SIP
  • LPA second set of bars with LPA concentrations ranging from 0.26-26
  • LPA is the negative control.
  • the instant invention is based on the discovery of a cDNA molecule encoding human GAVE8, a member of the G protein-coupled receptor superfamily, which binds to the endogenous ligand, sphingosine-l -phosphate (SIP, SPP-1 or SSP).
  • a nucleotide sequence encoding a human GAVE8 protein is shown in Figure 1 (SEQ ID NO:l).
  • An amino acid sequence of GAVE8 protein is shown in Figure 2 (SEQ ID NO:2).
  • the GAVE8 cDNA of Figure 1 (SEQ ID NO:l), which is approximately 2589 nucleotides long, including untranslated regions, encodes a protein having a molecular weight of approximately 41.8 kDa (excluding post-translational modifications).
  • GAVE8 is involved in a variety of disease states involving immune functions and various neurodegenerative diseases such as multiple sclerosis. Identification of GAVE8 in those tissues and cloning of the gene encoding GAVE8 provides a variety of therapeutic approaches to regulate GAVE8 expression and activity so as to provide therapeutic approaches to treating diseases involving GAVE8.
  • Human GAVE8 is related to the Edg family of molecules having certain conserved structural and functional features.
  • family when referring to the protein and nucleic acid molecules of the invention, is intended to mean two or more proteins or nucleic acid molecules having an overall common structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein.
  • family members can be naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin and a homologue of that protein of murine origin, as well as a second, distinct protein of human origin and a murine homologue of that protein.
  • Members of a family also may have common functional characteristics.
  • the Edg-1 receptor was isolated first by Hla & Maciag following stimulation of a human endothelial cell line by phorbol esters to identify genes implicated in cell differentiation (J Biol Chem (1990) 265:9308-9313). At that time, Edg-1 was classified as an orphan GPCR since no ligand was known to activate the receptor. Lee et al. later identified a serum-borne phospholipid called sphingosine-l -phosphate as the endogenous ligand for Edg-1 receptor (Science (1998) 279:1552-1555). The distinctive tissue distribution of GAVE8 expression directed efforts to the immune system and neural system.
  • the receptor also is conserved and is found in, for example, human, mouse and rat. Certain domains are well conserved across species.
  • GAVE8 also demonstrated modulation of expression associated with apoptosis. Thus, GAVE8 can be manipulated to induce or curtail programmed cell death.
  • a GAVE8 protein includes a third intracellular loop, domain, about amino acid 214 to about 252 having at least about 65% , preferably at least about 75% , and more preferably about 85% , 95% or 98% amino acid sequence identity to the third intracellular loop domain of SEQ ID NO:2.
  • amino acid residues herein means the amino acids occupy substantially the same position within a protein sequence when two or more sequences are aligned for analysis.
  • Preferred GAVE8 polypeptides of the instant invention have an amino acid sequence sufficiently identical to the third intracellular loop domain consensus amino acid sequence of SEQ ID NO:2.
  • the term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
  • amino acid or nucleotide sequences which contain a common structural domain having about 65% identity, preferably 75% identity, more preferably 85% , 95% or 98% identity are defined herein as sufficiently identical.
  • TM domains include, but are not limited to, the transmembrane (TM) domains (TM1 from about amino acid residue 38 to about 62; TM2 from about amino acid residue 70 to about 95; TM3 from about amino acid residue 112 to about 131 ; TM4 from about amino acid residue 151 to about 176; TM5 from about amino acid residue 193 to about 213; TM6 from about 253 to about 273; and TM7 from about amino acid residue 290 to about 310 as set forth in SEQ ID NO:2); cytoplasmic (intracellular loop) domains (from about amino acid residue 63 to about 69; from about amino acid residue 132 to about 150; from about amino acid residue 214 to about 252; and from about amino acid residue 31 1 to about 398 as set forth in SEQ ID NO:2); and extracellular domains (from about amino acid residue 1 to about 37; from about amino acid residue 96 to about 111; from about amino acid 177 to about 192; and from about amino acid residue 274 to about
  • GAVE8 or “functional activity of GAVE8” refers to an activity exerted by a GAVE8 protein, polypeptide or nucleic acid molecule on a GAVE8 responsive cell as determined in vivo or in vitro, according to standard techniques.
  • a GAVE8 activity can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction of the GAVE8 protein with a second protein.
  • a GAVE8 activity includes at least one or more of the following activities: (i) the ability to interact with proteins in the GAVE8 signaling pathway; (ii) the ability to interact with a GAVE8 ligand; and (iii) the ability to interact with an intracellular target protein.
  • a GAVE8 activity includes, but is not limited to, binding of sphingosine-l -phosphate (S-l-P), as may be determined by means such as fluorescent change (by FLIPR ® analysis) or binding of labeled S-l-P in cells transformed with an expression vector comprising SEQ ID NOT.
  • Another embodiment of the invention features isolated GAVE8 proteins and polypeptides having a GAVE8 activity.
  • nucleic acid molecules that encode GAVE8 proteins or biologically active portions thereof; as well as nucleic acid molecules sufficient for use as hybridization probes to identify GAVE8-encoding nucleic acids (e.g., GAVE8 mRNA) and fragments for use as PCR primers for the amplification or mutation of GAVE8 nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded.
  • an “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated GAVE8 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the instant invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOT, or a complement of any of those nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NOT as a hybridization probe, GAVE8 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a nucleic acid molecule of the invention can be amplified using cDN A, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • primers can comprise, but are not limited to 5'-CCATGGAGTCGGGGCTGC-3' (SEQ ID NO:3) and 5'-TCAGTCTGCAGCCGGTTC-3' (SEQ ID NO:4).
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • ohgonucleotides corresponding to GAVE8 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:l, or a portion thereof.
  • a nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence to thereby form a stable duplex.
  • the nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding GAVE8, for example, a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of GAVE8.
  • a fragment can comprise, but is not limited to, a region encoding amino acid residues 1-398 as set forth in SEQ ID NO:2.
  • the nucleotide sequence determined from the cloning of the human GAVE8 gene allows for the generation of probes and primers designed for use in identifying and/or cloning GAVE8 homologues in other cell types, e.g., from other tissues, as well as GAVE8 homologues from other mammals.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO: 1 or of a naturally occurring mutant of SEQ ID NO: 1.
  • Probes based on the human GAVE8 nucleotide sequence can be used to detect transcripts or genomic sequences encoding the similar or identical proteins.
  • the probe may comprise a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor.
  • a label group attached thereto e.g., a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which improperly express a GAVE8 protein, such as by measuring levels of a GAVE8-encoding nucleic acid in a sample of cells from a subject, e.g., detecting GAVE8 mRNA levels or determining whether a genomic GAVE8 gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of GAVE8” can be prepared by isolating a portion of SEQ ID NOT which encodes a polypeptide having a GAVE8 biological activity, expressing the encoded portion of GAVE8 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of GAVE8.
  • a nucleic acid fragment encoding a biologically active portion of GAVE8 includes a third intracellular loop domain, e.g., amino acid residues from about 214 to about 252 as set forth in SEQ ID NO:2.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO:l due to degeneracy of the genetic code and thus encode the same GAVE8 protein as that encoded by the nucleotide sequence shown in SEQ ID NOT .
  • GAVE8 nucleotide sequence shown in SEQ ID NOT
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of GAVE8 may exist within a population (e.g., the human population).
  • Such genetic polymorphism in the GAVE8 gene may exist among individuals within a population due to natural allelic variation.
  • An allele is one of a group of genes that occur alternatively at a given genetic locus.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a GAVE8 protein, preferably a mammalian GAVE8 protein.
  • allelic variant refers to a nucleotide sequence that occurs at a GAVE8 locus or to a polypeptide encoded by the nucleotide sequence.
  • Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. That can be carried out readily by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations in GAVE8 that are the result of natural allelic variation and that do not alter the functional activity of GAVE8 are intended to be within the scope of the invention.
  • nucleic acid molecules encoding GAVE8 proteins from other species which have a nucleotide sequence which differs from that of a human GAVE8, are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the GAVE8 cDNA of the invention can be isolated based on identity to the human GAVE8 nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule of the invention is at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000 or 1100 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence of SEQ ID NOT, or a complement thereof.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65% , 70% preferably 75% or greater) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found, for example, in “Current Protocols in Molecular Biology,” John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.
  • SSC sodium chloride/sodium citrate
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOT or the complement thereof corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of the GAVE8 sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NOT, thereby leading to changes in the amino acid sequence of the encoded GAVE8 protein, without altering the biological activity of the GAVE8 protein.
  • a "non-essential' amino acid residue is a residue that can be altered from the wild-type sequence of GAVE8 (e.g., the sequence of SEQ ID NO:2) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are not conserved or only semi-conserved among GAVE8 of various species may be non-essential for activity and thus would be likely targets for alteration.
  • amino acid residues that are conserved among the GAVE8 proteins of various species may be essential for activity and thus would not be likely targets for alteration.
  • another aspect of the invention pertains to nucleic acid molecules encoding GAVE8 proteins that contain changes in amino acid residues that are not essential for activity. Such GAVE8 proteins differ in amino acid sequence from SEQ ID NO:2 yet retain biological activity.
  • the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 87% identical, 90%, 93%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2.
  • An isolated nucleic acid molecule encoding a GAVE8 protein having a sequence which differs from that of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. Those families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g
  • a predicted nonessential amino acid residue in GAVE8 is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a GAVE8 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GAVE8 biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a mutant GAVE8 protein can be assayed for: (1) the ability to form proteimprotein interactions with proteins in the GAVE8 signaling pathway; (2) the ability to bind a GAVE8 ligand (e.g., SIP); or (3) the ability to bind to an intracellular target protein.
  • a mutant GAVE8 can be assayed for the ability to modulate cellular proliferation or cellular differentiation.
  • the instant invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire GAVE8 coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding GAVE8.
  • the noncoding regions (“5' and 3' untranslated or flanking regions") are the 5' and 3' sequences that flank the coding region and are not translated into amino acids.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of GAVE8 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of GAVE8 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GAVE8 mRNA, e.g., an oligonucleotide having the sequence 5'-GCAGCAGCCCCGACTCCATG-3' (SEQ ID NO:5) and 5'-CCATGGGCCGCGCCCCAAGG-3' (SEQ ID NO:6).
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be synthesized chemically using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives, phosphonate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, ⁇ -D-galactosylqueosine, inosine, N 6 -isopentenyladenine,
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a GAVE8 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracelluar concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong RNA polymerase (pol) II or pol III promoter are preferred.
  • An antisense nucleic acid molecule of the invention can be an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other (Gaultier et al., Nucleic Acids Res (1987)15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBS Lett 215:327-330).
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff et al., Nature (1988) 334:585-591)) can be used to catalytically cleave GAVE8 mRNA transcripts to thereby inhibit translation of GAVE8 mRNA.
  • a ribozyme having specificity for a GAVE8-encoding nucleic acid can be designed based on the nucleotide sequence of a GAVE8 cDNA disclosed herein (e.g., SEQ ID NOT).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a GAVE8-encoding mRNA. See, e.g., Cech et al., U.S. Patent No. 4,987,071; and Cech et al., U.S. Patent No. 5,116,742.
  • GAVE8 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., Science (1993) 261 : 141 1-1418.
  • the invention also encompasses nucleic acid molecules that form triple helical structures.
  • GAVE8 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the GAVE8 (e.g., the GAVE8 promoter and/or enhancers) to form triple helical structures that prevent transcription of the GAVE8 gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the GAVE8 e.g., the GAVE8 promoter and/or enhancers
  • the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (See Hyrup et al., Bioorganic & Medicinal Chemistry (1996) 4:5).
  • the terms "peptide nucleic acids” or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-OKeefe et al., Proc Natl Acad Sci USA (1996) 93:14670.
  • PNAs of GAVE8 can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of GAVE8 can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996), supra) or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996), supra).
  • PNAs of GAVE8 can be modified, e.g., to enhance their stability, specificity or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, Finn et al., Nucleic Acids Res (1996) 24(17):3357-63, Mag et al., Nucleic Acids Res (1989) 17:5973; and Peterser et al., Bioorganic Med Chem Lett (1975) 5:1119.
  • GAVE8 proteins and biologically active portions thereof, as well as polypeptide fragments suitable, for example, for use as immunogens to raise anti-GAVE8 antibodies.
  • native GAVE8 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • GAVE8 proteins are produced by recombinant DNA techniques.
  • Alternative to recombinant expression, a GAVE8 protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GAVE8 protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of GAVE8 protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • GAVE8 protein that is substantially free of cellular material includes preparations of GAVE8 protein having less than about 30% , 20% , 10% or 5% (by dry weight) of non-GAVE8 protein (also referred to herein as a "contaminating protein").
  • the GAVE8 protein or biologically active portion thereof is produced recombinantly, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20% , 10% or 5% of the volume of the protein preparation.
  • culture medium represents less than about 20% , 10% or 5% of the volume of the protein preparation.
  • GAVE8 protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of GAVE8 protein have less than about 30% , 20% , 10% or 5% (by dry weight) of chemical precursors or non-GAVE8 chemicals.
  • Biologically active portions of a GAVE8 protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the GAVE8 protein (e.g., the amino acid sequence shown in SEQ ID NO:2), which include fewer amino acids than the full length GAVE8 proteins, and exhibit at least one activity of a GAVE8 protein.
  • biologically active portions comprise a domain or motif with at least one activity of the GAVE8 protein.
  • a biologically active portion of a GAVE8 protein can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length.
  • Preferred biologically active polypeptides include one or more identified GAVE8 structural domains, e.g., the third intracellular loop domain (e.g., SEQ ID NO:2).
  • a preferred GAVE8 protein has the amino acid sequence shown of SEQ ID NO:2.
  • Other useful GAVE8 proteins are substantially identical to SEQ ID NO:2 and retain the functional activity of the protein of SEQ ID NO:2 yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • GAVE8 proteins and polypeptides possess at least one biological activity described herein.
  • a useful GAVE8 protein is a protein which includes an amino acid sequence at least about 88% , preferably 90% , 93% , 95% or 99% identical to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the GAVE8 proteins of SEQ ID NO:2.
  • the GAVE8 protein is a protein having an amino acid sequence 55% , 65% , 75% , 85% , 95% or 99% identical to the GAVE8 third intracellular loop domain (SEQ ID NO:2).
  • the GAVE8 protein retains a functional activity of the GAVE8 protein of SEQ ID NO:2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions then are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al., Proc Natl Acad Sci USA (1990) 87:2264, modified as in Karlin et al., Proc Natl Acad Sci USA (1993) 90:5873-5877.
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res (1997) 25:3389.
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers et al., CABIOS (1988) 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
  • GAVE8 chimeric or fusion proteins As used herein, a GAVE8 "chimeric protein” or “fusion protein” comprises a GAVE8 polypeptide operably linked to a non-GAVE8 polypeptide.
  • a “GAVE8 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to GAVE8
  • a non-GAVE8 polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially identical to the GAVE8 protein, e.g., a protein which is different from the GAVE8 protein and which is derived from the same or a different organism.
  • the GAVE8 polypeptide can correspond to all or a portion of a GAVE8 protein, preferably at least one biologically active portion of a GAVE8 protein.
  • the term "operably linked" is intended to indicate that the GAVE8 polypeptide and the non-GAVE8 polypeptide are fused in-frame to each other.
  • the non-GAVE8 polypeptide can be fused to the N-terminus or C-terminus of the GAVE8 polypeptide.
  • One useful fusion protein is a GST-GAVE8 fusion protein in which the GAVE8 sequences are fused to the C-terminus of a glutathione-S-transferase (GST) sequence. Such fusion proteins can facilitate the purification of recombinant GAVE8.
  • the third intracellular loop (IL3) of the instant invention (i.e., from about 214 to about 252 as set forth in SEQ ID NO:2) is fused with GST by PCR amplification of the IL3 and subcloning the product into a vector, such as, pGEX-2T.
  • the resulting construct can be introduced into a host cell (e.g., E. coli) and expression from said construct can be induced by an appropriate small molecule (e.g., isopropyl-1-thio- ⁇ -D-galactopyranoside) and subsequently purified (See, e.g., Lee et al., J Biol Chem (1996) 271(19):11272-1 1279).
  • a host cell e.g., E. coli
  • GAVE8 can be increased through use of a heterologous signal sequence.
  • the gp6 ® secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).
  • Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
  • the fusion protein is a GAVE8-immunoglobulin fusion protein in which all or part of GAVE8 is fused to sequences derived from a member of the immunoglobulin protein family.
  • the GAVE8-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a GAVE8 ligand and a GAVE8 protein on the surface of a cell, to thereby suppress GAVE 8 -mediated signal transduction in vivo.
  • the GAVE8-immunoglobulin fusion proteins can be used to affect the bioavailability of a GAVE8 cognate ligand. Inhibition of the GAVE8 ligand-GAVE8 interaction may be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g. promoting or inhibiting) cell survival.
  • the GAVE8-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-GAVE8 antibodies in a subject, to purify GAVE8 ligands and in screening assays to identify molecules which inhibit the interaction of GAVE8 with a GAVE8 ligand.
  • a GAVE8 chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example, by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which subsequently can be annealed and reamplified to generate a chimeric gene sequence (See e.g., Ausubel et al., supra).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which subsequently can be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a GAVE8-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GAVE8 protein.
  • the instant invention also pertains to variants of the GAVE8 proteins (i.e., proteins having a sequence that differs from that of the GAVE8 amino acid sequence). Such variants can function as either GAVE8 agonists (mimetics) or as GAVE8 antagonists. Variants of the GAVE8 protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the GAVE8 protein. An agonist of the GAVE8 protein can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the GAVE8 protein.
  • An antagonist of the GAVE8 protein can inhibit one or more of the activities of the naturally occurring form of the GAVE8 protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the GAVE8 protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the GAVE8 proteins.
  • Variants of the GAVE8 protein which function as either GAVE8 agonists (mimetics) or as GAVE8 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the GAVE8 protein for GAVE8 protein agonist or antagonist activity.
  • a variegated library of GAVE8 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of GAVE8 variants can be produced by, for example, enzymatically ligating a mixture of synthetic ohgonucleotides into gene sequences such that a degenerate set of potential GAVE8 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GAVE8 sequences therein.
  • a degenerate set of potential GAVE8 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GAVE8 sequences therein.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential GAVE8 sequences.
  • Methods for synthesizing degenerate ohgonucleotides are known in the art (See, e.g., Narang, Tetrahedron (1983) 39:3; Itakura et al., Ann Rev Biochem (1984) 53:323; Itakura et al., Science (1984) 198:1056; Ike et al., Nucleic Acid Res (1983) 11 :477).
  • libraries of fragments of the GAVE8 protein coding sequence can be used to generate a variegated population of GAVE8 fragments for screening and subsequent selection of variants of a GAVE8 protein.
  • a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of a GAVE8 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA which can include sense/antisense pairs from different nicked products, removing single-stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the GAVE8 protein.
  • REM Recursive ensemble mutagenesis
  • GAVE8 protein can be used as an immunogen to generate antibodies that bind GAVE8 using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length GAVE8 protein can be used or, alternatively, the invention provides antigenic peptide fragments of GAVE8 for use as immunogens.
  • the antigenic peptide of GAVE8 comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of GAVE8 such that an antibody raised against the peptide forms a specific immune complex with GAVE8.
  • an epitope may comprise an 8-mer comprising residues as set forth in SEQ ID NO:2.
  • epitopes encompassed by the antigenic peptide are regions of GAVE8 that are located on the surface of the protein, e.g., hydrophilic regions.
  • a hydrophobicity analysis of the human GAVE8 protein sequence indicates that the regions between about amino acids 1 and about 37, between about amino acids 96 and about 1 11, between about amino acids 177 and about 192 and between about amino acids 274 and about 290 of SEQ ID NO:2 are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • a GAVE8 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed GAVE8 protein or a chemically synthesized GAVE8 polypeptide.
  • the preparation further can include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic GAVE8 preparation induces a polyclonal anti-GAVE8 antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds GAVE8.
  • a molecule that specifically binds to GAVE8 is a molecule that binds GAVE8, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains GAVE8.
  • immunologically active portions of immunoglobulin molecules include F (a b) and F( at ,')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind GAVE8.
  • the term "monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GAVE8.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular GAVE8 protein with which it immunoreacts.
  • Polyclonal anti-GAVE8 antibodies can be prepared as described above by immunizing a suitable subject with a GAVE8 immunogen.
  • the anti-GAVE8 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized GAVE8.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against GAVE8 can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., Nature (1975) 256:495-497, the human B cell hybridoma technique (Kohler et al., Immunol Today (1983) 4:72), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, (1985), Alan R.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds GAVE8.
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the instant invention with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l , P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. Those myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion then are selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind GAVE8, e.g., using a standard ELISA assay.
  • PEG polyethylene glycol
  • a monoclonal anti-GAVE8 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with GAVE8 to thereby isolate immunoglobulin library members that bind GAVE8.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP ® Phage Display Kit, Catalog No. 240612).
  • recombinant anti-GAVE8 antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in 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; U.S. Patent No. 4,816,567; Europe Patent Application No.
  • Fully human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of GAVE8.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such an epitope, e.g., an antibody that inhibits GAVE8 activity, is identified.
  • the heavy chain and the light chain of the non-human antibody are cloned and used to create phage display Fab fragments.
  • the heavy chain gene can be cloned into a plasmid vector so that the heavy chain can be secreted from bacteria.
  • the light chain gene can be cloned into a phage coat protein gene so that the light chain can be expressed on the surface of phage.
  • a repertoire (random collection) of human light chains fused to phage is used to infect the bacteria that express the non-human heavy chain.
  • the resulting progeny phage display hybrid antibodies (human light chain/non-human heavy chain).
  • the selected antigen is used in a panning screen to select phage which bind the selected antigen.
  • human light chain genes are isolated from the selected phage which bind the selected antigen. These selected human light chain genes are then used to guide the selection of human heavy chain genes as follows. The selected human light chain genes are inserted into vectors for expression by bacteria. Bacteria expressing the selected human light chains are infected with a repertoire of human heavy chains fused to phage. The resulting progeny phage display human antibodies (human light chain/human heavy chain).
  • the selected antigen is used in a panning screen to select phage which bind the selected antigen.
  • the phage selected in that step display a completely human antibody that recognizes the same epitope recognized by the original selected, non-human monoclonal antibody.
  • the genes encoding both the heavy and light chains are isolated readily and can be manipulated further for production of human antibody. The technology is described by Jespers et al. (Bio/Technology (1994) 12:899-903).
  • An anti-GAVE8 antibody (e.g., monoclonal antibody) can be used to isolate GAVE8 by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-GAVE8 antibody can facilitate the purification of natural GAVE8 from cells and of recombinantly produced GAVE8 expressed in host cells.
  • an anti-GAVE8 antibody can be used to detect GAVE8 protein (e.g., in a cellular lysate or cell supernatant) to evaluate the abundance and pattern of expression of the GAVE8 protein.
  • Anti-GAVE8 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, galactosidase or acetylchohnesterase;
  • suitable prosthetic group complexes include streptavidin biotin and avidin/biotin;
  • suitable fluorescent materials include umbel liferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, given fluorescent protein or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and
  • suitable radioactive materials include 125 I, 13 I I, 35 S or 3 H.
  • vectors preferably expression vectors, containing a nucleic acid encoding GAVE8 (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • expression vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors).
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), that serve equivalent functions.
  • the recombinant expression vectors of the invention comprise nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. That means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of host cells to be used for expression, which is operably linked to the nucleic acid to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vivo transcription translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology Vol. 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in many types of host cells (e.g., tissue specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g., GAVE8 proteins, mutant forms of GAVE8, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of GAVE8 in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech inc.; Smith et al., Gene (1988) 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione 5-transferase (GST), maltose E binding protein or protein A, respectively, to the target recombinant protein.
  • GST glutathione 5-transferase
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene (1988) 69:301-315) and pET l id (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.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnl -lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). That viral polymerase is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California (1990) 185:119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., Nucleic Acids Res (1992) 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the GAVE8 expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSecl
  • GAVE8 can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., Mol Cell Biol (1983) 3:2156-2165) and the pVL series (Lucklow et al., Virology (1989) 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840) and pMT2PC (Kaufman et al., EMBO J (1987) 6:187-195).
  • the control functions of the expression vector often are provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art.
  • tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., Genes Dev (1987) 1 :268-277), lymphoid-specific promoters (Calame et al., Adv Immunol (1988) 43:235-275), in particular promoters of T cell receptors (Winoto et al., EMBO J (1989) 8:729-733) and immunoglobulins (Banerji et al., Cell (1983) 33:729-740; Queen et al., Cell (1983) 33:741-748), neuron-specific promoters (e.g., the 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 (
  • Patent No. 4,873,316 and European Application Publication No. 264,166 Developmentally-regulated promoters also are encompassed, for example the murine hox promoters (Kessel et al., Science (1990) 249:374-379) and the ⁇ -fetoprotein promoter (Campes et al., Genes Dev (1989) 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to GAVE8 mRNA. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but still are included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • GAVE8 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection or electroporation.
  • foreign nucleic acid e.g., DNA
  • a host cell including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection or electroporation.
  • a gene that encodes a selectable marker e.g., for resistance to antibiotics
  • Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding GAVE8 or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) GAVE8 protein.
  • the invention further provides methods for producing GAVE8 protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding GAVE8 has been introduced) in a suitable medium such that GAVE8 protein is produced.
  • the method further comprises isolating GAVE8 from the medium or the host cell.
  • the host cells of the invention also can be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which GAVE8-coding sequences have been introduced. Such host cells then can be used to create non-human transgenic animals in which exogenous GAVE8 sequences have been introduced into the genome or homologous recombinant animals in which endogenous GAVE8 sequences have been altered. Such animals are useful for studying the function and/or activity of GAVE8 and for identifying and/or evaluating modulators of GAVE8 activity.
  • a "transgenic animal” preferably is a mammal, more preferably, a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” preferably is a mammal, more preferably, a mouse, in which an endogenous GAVE8 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing GAVE8-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the GAVE8 cDNA sequence e.g., that of SEQ ID NO:l, can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of the human GAVE8 gene such as a mouse GAVE8 gene, can be isolated based on hybridization to the human GAVE8 cDNA and used as a transgene.
  • Intronic sequences and polyadenylation signals also can be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the GAVE8 transgene to direct expression of GAVE8 protein to particular cells.
  • Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, are conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals.
  • a transgenic founder animal then can be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a transgene encoding GAVE8 further can be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of a GAVE8 gene (e.g., a human or a non-human homolog of the GAVE8 gene, e.g., a murine GAVE8 gene) into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GAVE8 gene.
  • a GAVE8 gene e.g., a human or a non-human homolog of the GAVE8 gene, e.g., a murine GAVE8 gene
  • the vector is designed such that, on homologous recombination, the endogenous GAVE8 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" animal).
  • the vector can be designed such that, on homologous recombination, the endogenous GAVE8 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GAVE8 protein).
  • the altered portion of the GAVE8 gene is flanked at the 5' and 3' ends by additional nucleic acid of the GAVE8 gene to allow for homologous recombination to occur between the exogenous GAVE8 gene carried by the vector and an endogenous GAVE8 gene in an embryonic stem cell.
  • the additional flanking GAVE8 nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GAVE8 gene has homologously recombined with the endogenous GAVE8 gene are selected (See, e.g., Li et al., Cell (1992) 69:915).
  • the selected cells then are injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (See, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL, Oxford, (1987) pp.1 13-152).
  • a chimeric embryo then can be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system For a description of the cre/loxP recombinase system, see, e.g., Lakso et al., Proc Natl Acad Sci USA (1992) 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorrnan et al., Science (1991) 251 351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein also can be produced 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.
  • a cell e.g., a somatic cell
  • the quiescent cell then can be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte then is cultured such that it develops to morula or blastocyte and then transferred to a pseudopregnant female foster animal.
  • the offspring borne of that female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds also can be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with the intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • Acidity (pH) can be adjusted with acids or bases, such as HC1 or NaOH.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ® (BASF; Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringabihty exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyetheylene glycol and the like) and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a GAVE8 protein or anti-GAVE8 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a GAVE8 protein or anti-GAVE8 antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions also can be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally, swished and expectorated or swallowed.
  • compositions can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration also can be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants generally are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels or creams, as generally known in the art.
  • the compounds also can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. The materials also can be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) also can be used as pharmaceutically acceptable carriers. Those can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
  • Dosage unit form refers to physically discrete units suited to unitary dosages, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g., 0.1 to 20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful.
  • the progress of the therapy is monitored easily by conventional techniques and assays.
  • An exemplary dosing regimen is disclosed in WO 94/04188.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent No.
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack or dispenser, together with instructions for administration.
  • the nucleic acid molecules, proteins, protein homologues and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology); c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials and pharmacogenomics); and d) methods of treatment (e.g., therapeutic and prophylactic).
  • a GAVE8 protein interacts with other cellular proteins and can thus be used for (i) regulation of cellular proliferation; (ii) regulation of cellular differentiation; and (iii) regulation of cell survival.
  • the isolated nucleic acid molecules of the invention can be used to express GAVE8 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect GAVE8 mRNA (e.g., in a biological sample) or a genetic lesion in a GAVE8 gene, and to modulate GAVE8 activity.
  • the GAVE8 proteins can be used to screen drugs or compounds which modulate the GAVE8 activity or expression as well as to treat disorders characterized by insufficient or excessive production of GAVE8 protein or production of GAVE8 protein forms which have decreased or aberrant activity compared to GAVE8 wild-type protein.
  • the anti-GAVE8 antibodies of the invention can be used to detect and to isolate GAVE8 proteins and to modulate GAVE8 activity.
  • the invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to GAVE8 proteins or have a stimulatory or inhibitory effect on, for example, GAVE8 expression or GAVE8 activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to GAVE8 proteins or have a stimulatory or inhibitory effect on, for example, GAVE8 expression or GAVE8 activity.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a GAVE8 protein or polypeptide or biologically active portion thereof.
  • the test compounds of the instant invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des (1997) 12:145).
  • GAVE8 ligand is SIP
  • SIP can be investigated to determine the particular portion thereof that engages GAVE8, practicing known methods. That particular region can be synthesized practicing known biosynthetic methods, combining carbohydrate synthesis and enzymatic reactions, for example. That portion of SIP is equivalent to an "epitope.”
  • the GAVE8 epitope can be modified using other monomers or non-carbohydrate moieties to yield modified epitopes with enhanced properties, such as serum half-life, binding constant with GAVE8 and so on. The suitability of any one epitope variant can be determined practicing the binding and screening assays taught herein.
  • an assay is a cell-based assay in which a cell that expresses a membrane-bound form of GAVE8 protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a GAVE8 protein determined.
  • the cell for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the GAVE8 protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the GAVE8 protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, 14 C or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be labeled enzymatically with, for example, horseradish peroxidase, alkaline phosphatase or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of GAVE8 protein, or a biologically active portion thereof, on the cell surface with a known compound which binds GAVE8 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a GAVE8 protein, wherein determining the ability of the test compound to interact with a GAVE8 protein comprises determining the ability of the test compound to preferentially bind to GAVE8 or a biologically active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of GAVE8 protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GAVE8 protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of GAVE8 or a biologically active portion thereof can be accomplished, for example, by determining the ability of the GAVE8 protein to bind to or interact with a GAVE8 target molecule.
  • a "target molecule” is a molecule with which a GAVE8 protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a GAVE8 protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • a GAVE8 target molecule can be a non-GAVE8 molecule or a GAVE8 protein or polypeptide of the instant invention.
  • a GAVE8 target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a membrane-bound GAVE8 molecule) through the cell membrane and into the cell.
  • the target for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with GAVE8.
  • GAVE1 is made to signal constitutively using known techniques, see, for example WO 00/22131 and WO 00/22129, expressed in a target cell as taught herein, and then the cell is exposed to various candidate modulators to determine if signaling activity, the monitoring of which is described herein, is enhanced, revealing a candidate agonist, or diminished, revealing a candidate antagonist, or if activity is reduced below baseline levels, a candidate inverse agonist. Determining the ability of the GAVE8 protein to bind to or to interact with a
  • GAVE8 target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the GAVE8 protein to bind to or to interact with a GAVE8 target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., to include, but not limited to, intracellular Ca2 + , diacyl glycerol and IP3), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a GAVE8-responsive regulatory element operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cellular differentiation or cell proliferation.
  • a reporter gene e.g., a GAVE8-responsive regulatory element operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase
  • detecting a cellular response for example, cellular differentiation or cell proliferation.
  • an assay of the instant invention is a cell-free assay comprising contacting a GAVE8 protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the GAVE8 protein or biologically active portion thereof. Binding of the test compound to the GAVE8 protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the GAVE8 protein or biologically active portion thereof with a known compound which binds GAVE8 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a GAVE8 protein, wherein determining the ability of the test compound to interact with a GAVE8 protein comprises determining the ability of the test compound to preferentially bind to
  • GAVE8 or a biologically active portion thereof, as compared to the known compound.
  • an assay is a cell-free assay comprising contacting GAVE8 protein or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GAVE8 protein or a biologically active portion thereof. Determining the ability of the test compound to modulate the activity of GAVE8 can be accomplished, for example, by determining the ability of the GAVE8 protein to bind to a GAVE8 target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of GAVE8 can be accomplished by determining the ability of the GAVE8 protein to further modulate a GAVE8 target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described previously.
  • the cell-free assay comprises contacting the GAVE8 protein or biologically active portion thereof with a known compound which binds GAVE8 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a GAVE8 protein, wherein determining the ability of the test compound to interact with a GAVE8 protein comprises determining the ability of the GAVE8 protein to preferentially bind to or modulate the activity of a GAVE8 target molecule.
  • the cell-free assays of the instant invention are amenable to use of both the soluble form or the membrane-bound form of GAVE8.
  • cell-free assays comprising the membrane-bound form of GAVE8
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X- 114, Thesit ® , isotridecypoly(ethylene glycol-ether) n ,
  • binding of a test compound to GAVE8, or interaction of GAVE8 with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase/GAVE8 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which then are combined with the test compound or the test compound and either the non-adsorbed target protein or GAVE8 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • glutathione Sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized microtitre plates which then are combined with the test compound or the test compound and either the non-adsorbed target protein or GAVE8 protein, and the mixture incubated under conditions conducive to complex formation (e
  • the complexes can be dissociated from the matrix and the level of GAVE8 binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices also can be used in the screening assays of the invention.
  • GAVE8 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated GAVE8 or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemicals).
  • antibodies reactive with GAVE8 or target molecules but which do not interfere with binding of the GAVE8 protein to a target molecule can be derivatized to the wells of the plate, and unbound target or GAVE8 trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the
  • GST-immobilized complexes include immunodetection of complexes using antibodies reactive with the GAVE8 or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the GAVE8 or target molecule.
  • modulators of GAVE8 expression are identified in a method in which a cell is contacted with a candidate compound and the expression of GAVE8 mRNA or protein in the cell is determined.
  • the level of expression of GAVE8 mRNA or protein in the presence of the candidate compound is compared to the level of expression of GAVE8 mRNA or protein in the absence of the candidate compound.
  • the candidate compound then can be identified as a modulator of GAVE8 expression based on that comparison. For example, when expression of GAVE8 mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GAVE8 mRNA or protein expression.
  • the candidate compound when expression of GAVE8 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GAVE8 mRNA or protein expression.
  • the level of GAVE8 mRNA or protein expression in the cells can be determined by methods described herein for detecting GAVE8 mRNA or protein.
  • the GAVE8 proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. 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.
  • GAVE8-binding proteins bind to or interact with GAVE8
  • GAVE8-binding proteins bind to or interact with GAVE8
  • GAVE8-binding proteins bind to or interact with GAVE8
  • GAVE8-binding proteins are also likely to be involved in the propagation of signals by the GAVE8 proteins as, for example, upstream or downstream elements of the GAVE8 pathway.
  • the invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • portions or fragments of the cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents.
  • the sequences can be used to: (i) map respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Those applications are described in the subsections below.
  • GAVE8 nucleic acid molecules described herein or fragments thereof can be used to map the location of GAVE8 genes on a chromosome.
  • the mapping of the GAVE8 sequences to chromosomes is an important first step in correlating the sequences with genes associated with disease. Briefly, GAVE8 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the GAVE8 sequences. Computer analysis of GAVE8 sequences can be used to select primers that do not span more than one exon in the genomic DNA, which could complicate the amplification process. The primers then can be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the GAVE8 sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, the cells gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow (due to lack of a particular selecting enzyme), but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes.
  • mammals e.g., human and mouse cells.
  • Somatic cell hybrids containing only fragments of human chromosomes also can be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes.
  • mapping strategies which can similarly be used to map a GAVE8 sequence to its chromosome include in situ hybridization (described in Fan et al., Proc Natl Acad Sci USA (1990) 87:6223-27), pre-screening with labeled flow-sorted chromosomes and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical, e.g., colcemid, which disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1 ,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to non-coding regions of the genes also can be used for mapping purposes. Some coding sequences are conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GAVE8 gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the GAVE8 sequences of the instant invention also can be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of personnel.
  • RFLP restriction fragment length polymorphism
  • genomic DNA of an individual is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • the method does not suffer from the current limitations of "Dog Tags" which can be lost, switched or stolen, making positive identification difficult.
  • the sequences of the instant invention are useful as additional DNA markers for RFLP (described in U.S. Patent No. 5,272,057).
  • sequences of the instant invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of a genome.
  • the GAVE8 sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. The primers then can be used to amplify genomic DNA and subsequently provide the sequence thereof.
  • Panels of corresponding DNA sequences from individuals, prepared in that manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the instant invention can be used to obtain such identification sequences from individuals and from tissue.
  • the GAVE8 sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of the sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • the noncoding sequences of SEQ ID NOT can provide positive individual identification with a panel of perhaps 10 to 1 ,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOT are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from GAVE8 sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • DNA-based identification techniques also can be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva or semen, found at a crime scene. The amplified sequence then can be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the instant invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e., another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e., another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NOT are particularly appropriate for that use as greater numbers of polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using that technique.
  • polynucleotide reagents include the GAVE8 sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NOT having a length of at least 20 or 30 bases.
  • the GAVE8 sequences described herein can be used further to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. That can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such GAVE8 probes can be used to identify tissue by species and/or by organ type.
  • the reagents e.g., GAVE8 primers or probes can be used to screen tissue culture for contamination (i.e., screen for the presence of a mixture of different types of cells in a culture).
  • the instant invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics and monitoring clinical trails are used for prognostic (predictive) pu ⁇ oses to treat thereby an individual prophylactically.
  • diagnostic assays for determining GAVE8 protein and/or nucleic acid expression as well as GAVE8 activity, in the context of a biological sample (e.g., blood, urine, feces, serum, cells, tissue) to determine thereby whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant GAVE8 expression or activity.
  • a biological sample e.g., blood, urine, feces, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with GAVE8 protein, nucleic acid expression or activity. For example, mutations in a GAVE8 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with GAVE8 protein, nucleic acid expression or activity.
  • Another aspect of the invention provides methods for determining GAVE8 protein, nucleic acid expression or GAVE8 activity in an individual to select thereby appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent).
  • agents e.g., drugs
  • Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of GAVE8 in clinical trials.
  • agents e.g., drugs or other compounds
  • An exemplary method for detecting the presence or absence of GAVE8 in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting
  • GAVE8 protein or nucleic acid that encodes GAVE8 protein such that the presence of GAVE8 is detected in the biological sample.
  • a preferred agent for detecting GAVE8 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to GAVE8 mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length GAVE8 nucleic acid, such as the nucleic acid of SEQ ID NOT or a portion thereof, such as an oligonucleotide of at least 15, 30,
  • a preferred agent for detecting GAVE8 protein is an antibody capable of binding to GAVE8 protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • the term, "labeled", with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect GAVE8 mRNA, protein or genomic DNA in a biological sample in vivo as well as in vitro.
  • in vivo techniques for detection of GAVE8 mRNA include Northern hybridization and in situ hybridization.
  • In vitro techniques for detection of GAVE8 protein include enzyme linked immunosorbent assay (ELISAs), Western blot, immunoprecipitation and immunofluorescence.
  • In vitro techniques for detection of GAVE8 genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of GAVE8 protein include introducing into a subject a labeled anti-GAVE8 antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting GAVE8 protein, mRNA or genomic DNA, such that the presence of GAVE8 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of GAVE8 protein, mRNA or genomic DNA in the control sample with the presence of GAVE8 protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of GAVE8 in a biological sample can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of GAVE8 (e.g., an immunological disorder).
  • the kit can comprise a labeled compound or agent capable of detecting GAVE8 protein or mRNA in a biological sample and means for determining the amount of GAVE8 in the sample (e.g., an anti-GAVE8 antibody or an oligonucleotide probe which binds to DNA encoding GAVE8, e.g., SEQ ID NOT).
  • Kits also can include instructions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of GAVE8, if the amount of GAVE8 protein or mRNA is above or below a normal level.
  • the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to GAVE8 protein; and, optionally, (2) a second, different antibody which binds to GA VE8 protein or the first antibody and is conjugated to a detectable agent.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody which binds to GA VE8 protein or the first antibody and is conjugated to a detectable agent.
  • the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a GAVE8 nucleic acid sequence or (2) a pair of primers useful for amplifying a GAVE8 nucleic acid molecule.
  • an oligonucleotide e.g., a detectably labeled oligonucleotide, which hybridizes to a GAVE8 nucleic acid sequence
  • a pair of primers useful for amplifying a GAVE8 nucleic acid molecule.
  • the kit also can comprise, e.g., a buffering agent, a preservative or a protein stabilizing agent.
  • the kit also can comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • the kit also can contain a control sample or a series of control samples that can be assayed and compared to the test sample contained.
  • Each component of the kit usually is enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of GAVE8.
  • the methods described herein furthermore can be utilized as diagnostic or prognostic assays to identify subjects having or are at risk of developing a disease or disorder associated with aberrant GAVE8 expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or is at risk of developing a disorder associated with GAVE8 protein, nucleic acid expression or activity, e.g., disorders of the immune system or neural system, such as an immune response associated with onset of multiple sclerosis.
  • the prognostic assays can be utilized to identify a subject having or is at risk for developing such a disease or disorder.
  • test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule or other drug candidate) to treat a disease or disorder associated with aberrant GAVE8 expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule or other drug candidate
  • such methods can be used to determine whether a subject can be treated effectively with a specific agent or class of agents (e.g., agents of a type that decrease GAVE8 activity).
  • the instant invention provides methods for determining whether a subject can be treated effectively with an agent for a disorder associated with aberrant GAVE8 expression or activity in which a test sample is obtained and GAVE8 protein or nucleic acid is detected (e.g., wherein the presence of GAVE8 protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant GAVE8 expression or activity).
  • the methods of the invention also can be used to detect genetic lesions or mutations in a GAVE8 gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding a GAVE8-protein, or the misexpression of the GAVE8 gene.
  • such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from a GAVE8 gene; 2) an addition of one or more nucleotides to a GAVE8 gene; 3) a substitution of one or more nucleotides of a GAVE8 gene; 4) a chromosomal rearrangement of a GAVE8 gene; 5) an alteration in the level of a messenger RNA transcript of a GAVE8 gene; 6) an aberrant modification of a GAVE8 gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a GAVE8 gene; 8) a non- wild-type level of a GAVE8-protein; 9) an allelic loss of a GAVE8 gene; and 10) an inappropriate post-translational modification of a GAVE8 protein.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (See, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (See, e.g., Landegran et al., Science (1988) 241 :1077-1080; and Nakazawa et al., Proc Natl Acad Sci USA (1994) 91 :360-364), the latter of which can be particularly useful for detecting point mutations in the GAVE8 gene (See, e.g., Abravaya et al., Nucleic Acids Res (1995) 23:675-682).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • the method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a GAVE8 gene under conditions such that hybridization and amplification of the GAVE8 gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli et al., Proc Natl Acad Sci USA (1990) 87:1874-1878), transcriptional amplification system (Kwoh et al., Proc Natl Acad Sci USA (1989) 86:1173-1177), Q- ⁇ Replicase (Lizardi et al., Bio/Technology (1988) 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.
  • the detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a GAVE8 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA are isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicate mutations in the sample DNA.
  • sequence-specific ribozymes see, e.g., U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in GAVE8 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of ohgonucleotides probes (Cronin et al., Human Mutation (1996) 7:244-255; Kozal et al., Nature Medicine (1996) 2:753-759).
  • genetic mutations in GAVE8 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
  • That step allows the identification of point mutations. That step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the GAVE8 gene and to detect mutations by comparing the sequence of the sample GAVE8 with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim & Gilbert (Proc Natl Acad Sci USA (1977) 74:560) or Sanger (Proc Natl Acad Sci USA (1977) 74:5463). It also is contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Bio/Techniques (1995) 19:448), including sequencing by mass spectrometry (See, e.g., PCT Publication No.
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the GAVE8 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al., Science (1985) 230: 1242).
  • the technique of "mismatch cleavage" entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type GAVE8 sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions.
  • DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine to digest mismatched regions. After digestion of the mismatched regions, the resulting material then is separated by size on denaturing polyacrylamide gels to determine the site of mutation.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in GAVE8 cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al., Carcinogenesis (1994) 15:1657-1662).
  • a probe based on a GAVE8 sequence e.g., a wild-type GAVE8 sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in GAVE8 genes.
  • single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al., Proc Natl Acad Sci USA (1989) 86:2766; See also Cotton, Mutat Res (1993) 285:125-144; Hayashi, Genet Anal Tech Appl (1992) 9:73-79). Single-stranded DNA fragments of sample and control GAVE8 nucleic acids will be denatured and allowed to renature.
  • SSCP single strand conformation polymo ⁇ hism
  • the secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al., Trends Genet (1991) 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature (1985) 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum et al., Biophys Chem (1987) 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al., Nature (1986) 324:163); Saiki et al, Proc Natl Acad Sci USA (1989) 86:6230).
  • Such allele-specific ohgonucleotides are hybridized to PCR-amplified target DNA or a number of different mutations when the ohgonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • allele-specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention.
  • Ohgonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al., Nucleic Acids Res (1989) 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner, Tibtech (1993) 1 1 :238).
  • amplification also may be performed using Taq ligase for amplification (Barany, Proc Natl Acad Sci USA (1991) 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be used conveniently, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a GAVE8 gene.
  • any cell type or tissue where GAVE8 is expressed may be utilized in the prognostic assays described herein.
  • Agents, or modulators which have a stimulatory or inhibitory effect on GAVE8 activity can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., including, but not limited to, immune responses associated with multiple sclerosis) associated with GAVE8 activity.
  • disorders e.g., including, but not limited to, immune responses associated with multiple sclerosis
  • the pharmacogenomics i.e., the study of the relationship between the genotype of an individual and the response of that individual to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the genotype of an individual. Such pharmacogenomics further can be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of GAVE8 protein, expression of GAVE8 nucleic acid or mutation content of GAVE8 genes in an individual can be determined thereby to select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder, Clin Chem (1997) 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes, CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the polymo ⁇ hisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and the poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by the CYP2D6-formed metabolite, mo ⁇ hine. The other extreme is the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of GAVE8 protein, expression of GAVE8 nucleic acid or mutation content of GAVE8 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic alleles encoding the drug-metabolizing enzymes to the identification the drug responsiveness phenotype of an individual. That knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a GAVE8 modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • GAVE8 e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • agents e.g., drugs, compounds
  • GAVE8 e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • the effectiveness of an agent, as determined by a screening assay as described herein, to increase GAVE8 gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting decreased GAVE8 gene expression, protein levels or protein activity.
  • the effectiveness of an agent, as determined by a screening assay, to decrease GAVE8 gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting increased GAVE8 gene expression, protein levels or protein activity.
  • GAVE8 expression or activity and preferably, that of other genes that have been implicated in, for example, a cellular proliferation disorder can be used as a marker of the immune responsiveness of a particular cell.
  • genes, including GAVE8, that are modulated in cells by treatment with an agent e.g., compound, drug or small molecule
  • an agent e.g., compound, drug or small molecule
  • GAVE8 activity e.g., as identified in a screening assay described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of GAVE8 and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of GAVE8 or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, the response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the instant invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule or other drug candidate identified by the screening assays described herein) comprising the steps of: (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a GAVE8 protein, mRNA or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the GAVE8 protein, mRNA or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the GAVE8 protein, mRNA or genomic DNA in the pre-administration sample with the GAVE8 protein, mRNA or genomic DNA in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g.,
  • increased administration of the agent may be desirable to increase the expression or activity of GAVE8 to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of GAVE8 to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • the instant invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant GAVE8 expression or activity.
  • disorders include, but are not limited to, immune responses associated with multiple sclerosis.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant GAVE8 expression or activity, by administering to the subject an agent that modulates GAVE8 expression or at least one GAVE8 activity.
  • Subjects at risk for a disease that is caused or contributed to by aberrant GAVE8 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the GAVE8 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in progression.
  • a GAVE8 agonist or GAVE8 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of GAVE8 protein activity associated with the cell.
  • An agent that modulates GAVE8 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a GA VE8 protein, a peptide, a GAVE8 peptidomimetic or other small molecule.
  • the agent can be an agonist, inverse agonist or antagonist.
  • the agent stimulates one or more of the biological activities of GAVE8 protein.
  • Examples of such stimulatory agents include active GAVE8 protein and a nucleic acid molecule encoding GAVE8 that has been introduced into the cell.
  • the agent inhibits one or more of the biological activities of GAVE8 protein.
  • Examples of such inhibitory agents include antisense GAVE8 nucleic acid molecules and anti-GAVE8 antibodies.
  • the modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the instant invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a GAVE8 protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein) or combination of agents that modulates (e.g., upregulates or downregulates) GAVE8 expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a GAVE8 protein or nucleic acid molecule as therapy to compensate for reduced or aberrant GAVE8 expression or activity.
  • Stimulation of GAVE8 activity is desirable in situations in which GAVE8 is abnormally downregulated and/or in which increased GAVE8 activity is likely to have a beneficial effect. Conversely, inhibition of GAVE8 activity is desirable in situations in which GAVE8 is abnormally upregulated and/or in which decreased GAVE8 activity is likely to have a beneficial effect.
  • a partial human GAVE8 coding region (AC026510) was identified from a human genome database by the FASTA algorithm. Primers specific to the human GAVE8 coding region: 5' CGCGGTGCGCGCTACCAG 3' (SEQ ID NO:7) and 5' GCGCCTGCCAGCAGATCC 3' (SEQ ID NO:8) were used for PCR screening to isolate clones from a human brain cDNA library. The human cDNA library with primary recombinant clones was subdivided into 90 pools. Each pool containing about 3 x 10 4 clones was loaded into wells of a 96-well PCR plate for PCR amplification.
  • PCR protocol 94° C, hold for 3 min.; 40 cycles of 94° C for 30 seconds, 52° C for 30 seconds and 68° C for 45 seconds.
  • the PCR products from each well of the 96-well PCR plate were checked by electrophoresis on 2% agarose gels.
  • the subpools showed the expected 185 bp PCR fragment of potential GAVE8 clones.
  • Positive subpools subsequently were diluted to another 90 pools for further PCR screening. A limited number of colonies from the second round of positive subpools were plated on agar plates and positive plasmids were verified by PCR and were subjected to DNA sequencing analysis.
  • hGAVE8 To provide significant quantities of hGAVE8 for further experiments, the cDNA encoding hGAVE8 was cloned into an expression vector and transfected into HEK293 cells.
  • HEK293 cells were plated in a six-well 35 mm tissue culture plate (3 x 10 5 HEK293 cells per well (ATCC Catalog
  • the cells then were incubated at 37° C in a C0 2 incubator until the cells were 50-80% confluent.
  • the cloned cDNA nucleic acid sequence of hGAVE8 was inserted using the procedure described above in a pcDNA 3.1 cloning vector (Invitrogen,
  • transfection mixture For each transfection (six transfections in a six-well plate), 0.8 ml of serum-free F12 HAM media was added to the solution containing the DNA-lipid complexes (0.2 ml total volume) and mixed gently. The resulting mixture (hereinafter the "transfection mixture") then was overlaid (0.8 ml + 0.2 ml) onto the rinsed cells. No anti-bacterial reagents were added. The cells then were incubated with the lipid-DNA complexes for 16 hours at 37° C in a C0 2 incubator to allow for transfection.
  • F12 HAM media containing 10% fetal bovine serum was overlaid onto the cells without first removing the transfection mixture.
  • the media overlaying the cells was aspirated.
  • Cells then were washed with PBS pH 2-4 (Gibco/BRL Catalog No. 10010-023) and PBS was replaced with F12 HAM media containing 5% serum ("selective media").
  • the cells were diluted ten-fold into the selective medium containing the antibacterial agent genetecin at 400 ⁇ g/ml (Life Technologies, Catalog No. 1181 1).
  • hGAVE8 is artificially coupled to a G a ⁇ 5 signaling package.
  • Activation of the G q mechanism stimulates the release of Ca from sarcoplasmic reticulum vesicles within the cell.
  • the Ca 2+ is released into the cytoplasm where it can be detected using Ca 2+ chelating dyes.
  • a Fluorometric Imaging Plate Reader or FLIPR apparatus (Molecular Devices) is used to monitor any resulting changes in fluorescence using a luciferase reporting gene system. The activity of an agonist is reflected by an increase in fluorescence.
  • HEK293 cells expressing hGAVE8 are pre-engineered to express an indiscriminate form of Gq protein (G aq ⁇ 5 ). To prepare such cells, G ⁇ ⁇ 6 -coupled HEK293 cells are used and the protocol in Example 2 followed to facilitate expression ofhGAVE8 in the cells.
  • the cells are maintained in log phase of growth at 37° C and 5% CO 2 in F12 HAM media (Gibco/BRL, Catalog No. 11765-054) containing 10% fetal bovine serum, 100 IU/ml penicillin (Gibco/BRL, Catalog No. 15140-148), 100 ⁇ g/ml streptomycin (Catalog No. 15140-148, Gibco/BRL), 400 ⁇ g/ml geneticin (G418) (Gibco/BRL, Catalog No. 10131-035) and 200 ⁇ g/ml zeocin (Invitrogen, Catalog No. R250-05).
  • F12 HAM media Gibco/BRL, Catalog No. 11765-054
  • penicillin Gibco/BRL
  • streptomycin Catalog No. 15140-148, Gibco/BRL
  • G418 Gibco/BRL, Catalog No. 10131-035
  • 200 ⁇ g/ml zeocin Invitrogen, Catalog No. R250-
  • HEK293 cells Twenty-four hours prior to an assay, 12,500 cells/well of the HEK293 cells are plated onto 384-well clear-bottomed assay plates with a well volume of 50 ⁇ l (Greiner/Marsh, Catalog No. N58102) using a 96/384 Multidrop device (Labsystems, Type 832). The cells are incubated at 37° C in humidified 5% C0 2 incubator (Forma Scientific C0 2 water jacketed incubator Model 3110).
  • the following stock solutions are prepared: a 1 M stock solution of Hepes (pH 7.5) (Gibco/BRL, Catalog No. 15630-080); a 250 mM stock solution of probenicid (Sigma, Catalog No. P8761) made in 1 N NaOH; and a 1 mM stock solution of Fluo 4-AM Dye (Molecular Probes, Catalog No. FI 4202) made in DMSO (Sigma D2650).
  • Reaction buffer is prepared with 1000 ml Hank's Balanced Salt Solution (Fisher/Mediatech, Catalog No. MT21023), 20 ml of the 1 M Hepes stock solution and 10 ml of the 250 mM probenicid stock solution.
  • the CCD camera (Princeton Instruments) of the FLIPR ® II (Molecular Devices) instrument is set at an f-stop of 2.0 and an exposure of 0.4 seconds.
  • the camera is used to monitor cell plates for accuracy of dye loading.
  • a compound library containing possible agonists will be tested at 10 ⁇ M concentration in physiological salt buffer. Changes in fluorescence are measured for 10 seconds prior to compound addition. After the addition of the compound, fluorescence is measured every second for the first minute followed by exposures taken every six seconds for a total experimental analysis time of three minutes. Five ⁇ l aliquots of the 100 ⁇ M stock compound are added after the tenth scan, giving a final compound concentration on the cells of 10 ⁇ M. The maximum fluorescence changes for the first 80 scans is recorded as a measure of agonist activity and compared to the maximum fluorescence change induced by 10 ⁇ M ATP (Sigma A9062).
  • hGAVE8 is artificially coupled to a G q system.
  • a FLIPR ® apparatus will be used to monitor any resulting changes in fluorescence.
  • the activity of an antagonist is reflected by any decrease in fluorescence.
  • HEK293 cells expressing hGAVE8 are pre-engineered to express an indiscriminate form of Gq protein (G aq i 5 ), as described above in Example 3.
  • the cells are maintained in log phase of growth at 37° C and 5% CO 2 in F12 HAM media (Gibco/BRL, Catalog No. 11765-054) containing 10% fetal bovine serum, lOO IU/ml penicillin (Gibco/BRL, Catalog No. 15140-148), 100 ⁇ g/ml streptomycin (Catalog No. 15140-148, Gibco/BRL), 400 ⁇ g/ml geneticin (G418) (Gibco/BRL, Catalog No.
  • the following stock solutions will be prepared: a 1 M stock solution of Hepes (pH 7.5) (Gibco/BRL, Catalog No. 15630-080); a 250 mM stock solution of probenicid (Sigma, Catalog No. P8761) made in 1 N NaOH; a 1 mM stock solution of Fluo 4-AM Dye (Molecular Probes, Catalog No. F 14202) made in DMSO (Sigma D2650); and a 480 nM stock solution of SPP-1 (BioMol).
  • Reaction buffer is prepared with 1000 ml Hank's Balanced Salt Solution (Fisher/Mediatech, Catalog No.
  • HEK293 cell lines overexpressing hGAVE8 are harvested by incubation in phosphate-buffered saline (10 ml) containing 1 mM EDTA. The cells are washed further 3 times in phosphate-buffered saline containing 1 mM EDTA (10 ml) prior to resuspension in 5 ml of Buffer A (50 mM Tris-HCl (pH 7.8) (Sigma T6791), 5 mM MgCl 2 (Sigma M8266), and 1 mM EGTA (Sigma 0396).
  • Buffer A 50 mM Tris-HCl (pH 7.8) (Sigma T6791), 5 mM MgCl 2 (Sigma M8266), and 1 mM EGTA (Sigma 0396).
  • the cells then are disrupted with a tissue homogenizer (Polytron, Kinemetica, Model PT 10/35) for 1 minute.
  • tissue homogenizer Polytron, Kinemetica, Model PT 10/35) for 1 minute.
  • the resulting homogenate is centrifuged in a Sorvall Instruments RC3B refrigerated centrifuge at 49,000 x g at 4° C for 20 minutes.
  • the resulting pellet is resuspended in 25 ml of Buffer A and the centrifugation step is repeated three times. Following the final centrifugation, the pellet is again resuspended in 5 ml of Buffer A, aliquoted and stored at -70° C.
  • a receptor binding assay using the membrane fraction and radiolabeled sphingosine-l -phosphate (or SPP-1) as a tracer is performed.
  • the assay is performed in a 96-well plate (Beckman Instruments).
  • the binding reaction consisted of 18 ⁇ g of the HEK293 cell preparation in the presence of radioactive SPP-1 (0.01 nM-25 nM) in a final volume of 0.2 ml of Buffer A containing 0.1% bovine serum albumin (Sigma, Catalog No. 34287) (See Im et al., J Biol Chem (2000) 275(19):14281-14286).
  • the reaction is incubated for 1 hour at room temperature.
  • the reaction is terminated by filtration through Whatman GF/C filters on a multichannel harvester (Brandell) which is pretreated with 0.3% polyethyleneimine (Sigma, Catalog No. P3143) and 0.1% bovine serum albumin (BSA) for 1 hour. The mixture is applied to the filter and incubated for one hour. The filters are washed 6 times with 1 ml of ice cold 50 mM Tris-HCl pH 7.6. Specific binding is calculated based on the difference between total binding and non-specific binding (background) for each tracer concentration by measuring the radioactivity.
  • Northern blot analysis is performed on RNA derived from several human tissue samples to determine whether the tissues express the hGAVE8 receptor gene.
  • Commercially available filters containing RNA's from multiple human tissues can be used, such as that from Clontech.
  • the probe used is P 32 -labeled full length I1GAVE8 cDNA.
  • P32-labeled hGAVE8 cDNA is prepared as follows. Twenty-five ng of hGAVE8 cDNA prepared as described above is resuspended to 45 ⁇ l of 10 mM Tris-HCl, pH 7.5; 1 mM EDTA in a microfuge tube and heated at 95° C for 5 minutes. The tube then is chilled on ice for another 5 minutes. Following chilling, the mixture contained in the tube is resuspended with the 45 ⁇ l GAVE8 cDNA and buffer as described above and mixed with RTS Rad Prime Mix (supplied with the RTS Rad Prime DNA-labeling System) (Life Technologies, Catalog No. 10387-017).
  • RTS Rad Prime Mix supplied with the RTS Rad Prime DNA-labeling System
  • Cells or tissues are directly lysed in a culture dish by adding 1 ml of Trizol Reagent (Life Technologies, Catalog No. 15596). The cell lysate then is passed through a pipette several times to homogenize the lysate (cell lysate is subsequently is transferred to a tube). Following homogenization, the lysate is incubated for 5 minutes at 30° C to permit the complete dissociation of nucleoprotein complexes. Following incubation, 0.2 ml of chloroform (Sigma, Catalog No. C53 12) per 1 ml of Trizol Reagent is added to the lysate and the tube is shaken vigorously for 15 seconds. The lysate then is incubated at 30° C for 3 minutes.
  • Trizol Reagent Life Technologies, Catalog No. 15596
  • RNAase-free water (Life Technologies, Catalog No. 10977-015).
  • An agarose gel is prepared by melting 2 g of agarose (Sigma, Catalog No. A0169) in water, 5X formaldehyde gel-running buffer (See below for description) and 2.2 M formaldehyde (Sigma, Catalog No. P82031).
  • Samples for gel electrophoresis are prepared as follows:
  • the samples are incubated for 15 minutes at 65° C and then chilled on ice.
  • RNA samples from various organs can be purchased.
  • Commercially available (Clontech) hybridization filters containing a plurality of RNA samples from various organs can be purchased.
  • the gel is pre-run for 5 minutes at 5 V/cm. Following the pre-run, the samples were loaded onto the gel. The gel then is run at 4 V/cm while submerged in formaldehyde gel-running buffer. The buffer is changed at 2 hours into the run.
  • RNA is transferred from the agarose gel to a nitrocellulose filter (Schleicher & Schuell Inc, Catalog No. 74330-026) using the protocol described in Sambrook et al., eds. (in Molecular Cloning: A Laboratory Manual, volume 1, pp.7.46-7.51, Cold Spring Harbor Laboratory Press (1989)).
  • Clontech ExpressHyb hybridization solution (Clontech, Catalog No. 8015-1) is incubated at 68° C for 2 hours. Following incubation, 15 ml of the warmed hybridization solution is poured onto the multiple tissue sample Northern (MTN) membrane. The MTN membrane is left soaking in the hybridization solution at 68° C while shaking. After 1 hour has elapsed, the hGAVE8 cDNA probe, which had been previously denatured by boiling at 95° C for 5 minutes, is added at a concentration of 10 6 counts/ml. The incubation of the hybridization solution covering the gel at 68° C then is continued for 2 hours and up through overnight while shaking.
  • MTN multiple tissue sample Northern
  • Clontech Wash Solution 1 (2X SSC; 0.05% SDS)
  • Clontech Wash Solution 2 (0.1X SSC; 0.1% SDS) then is warmed at 60° C for 1 hour.
  • the membrane then is washed 3 consecutive times with Clontech Wash Solution 2 (0.1X SSC; 0.1% SDS) by dipping the membrane into 15 ml of solution while shaking at 60° C temperature for 60 minutes.
  • the wash solution is changed every 15 minutes.
  • the membrane is exposed to Kodak X-OMAT AR (Kodak, Catalog No. 165 1579) film overnight at -70° C with intensifying screens.
  • Kodak X-OMAT AR Kodak, Catalog No. 165 1579
  • Results hGAVE8 is expressed at high levels in the human spleen, brain and PBL.
  • the size of the unique transcript is about 2.4 kb.
  • TaqMan ® or real time RT-PCR is a powerful tool for detecting messenger RNA in samples.
  • the technology exploits the 5' nuclease activity of AmpliTaq Gold ® DNA polymerase to cleave a TaqMan" probe during PCR.
  • the TaqMan ® probe contains a reporter dye (in the experiments: 6-FAM (6-carboxyfluorescein)) at the 5'-end of the probe and a quencher dye (in the experiments: TAMRA (6-carboxy-N, N, N',
  • TaqMan ® probes are specifically designed to hybridize with the target cDNA of interest between the forward and the reverse primer sites.
  • the 3 '-end quencher dye suppresses the fluorescence of the 5'-end reporter dye.
  • the 5' ⁇ 3' activity of the AmpliTaq Gold ® DNA polymerase results in the cleavage of the probe between the 5'-end reporter dye and the 3'-end quencher dye resulting in the displacement of the reporter dye. Once displaced, the fluorescence of the reporter dye no longer is suppressed by the quencher dye.
  • the accumulation of PCR products made from the targeted cDNA template is detected by monitoring the increase in fluorescence of the reporter dye.
  • An ABI Prism Sequence detector system from Perkin Elmer Applied Biosystems (Model No. ABI7700) is used to monitor the increase of the reporter fluorescence during PCR.
  • the reporter signal is normalized to the emission of a passive reference.
  • RNA and poly A + RNA from several tissues are purchased from Clontech (See Tables 1 and 2 for catalog numbers).
  • RNA Five ⁇ g of total RNA is mixed with 2 ⁇ l (50 ng/ ⁇ l) of random hexamer primers
  • the mixture then is incubated for 65° C for 10 minutes.
  • the PCR and TaqMan ® assay are performed in a 96-well plate MicroAmp optical tube (Perkin Elmer, Catalog No. N801-0933).
  • a reaction mixture comprising 25 ⁇ l of TaqMan ® PCR Mixture (Perkin Elmer, Catalog No.
  • Results from the above TaqMan reactions are expressed relative to a tissue of arbitrary choice as fold regulation (for instance, value of the GAVE8 expression in the heart/value of the GAVE8 expression in the brain).
  • fold regulation for instance, value of the GAVE8 expression in the heart/value of the GAVE8 expression in the brain.
  • cerebellum is chosen as a reference to which the expression of GAVE8 is compared to and relative fold expression is deduced.

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Abstract

L'invention concerne de nouveaux polypeptides, des protéines et des molécules d'acide nucléique GAVE8. L'invention concerne également des protéines GAVE8 pleine longueur isolées, des protéines hybrides GAVE8 isolées, des peptides antigéniques et des anticorps anti-GAVE8. L'invention concerne également des molécules d'acide nucléique GAVE8, des vecteurs d'expression recombinés contenant cette molécule d'acide nucléique, des cellules hôtes dans lesquelles les vecteurs d'expression ont été introduits et des animaux transgénique non humains dans lesquels le gène GAVE8 a été introduit ou interrompu. L'invention concerne également des méthodes de diagnostic, de criblage et de thérapie faisant intervenir des compositions ou des molécules GAVE8 fixant GAVE8. L'invention concerne également des méthodes destinées à l'identification d'agonistes, d'antagonistes, d'agonistes inverses de GAVE8 et d'éléments analogues.
PCT/US2002/023208 2001-07-20 2002-07-19 Nouveau recepteur couple aux proteines g, gave8 WO2003008560A2 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
EP02747073A EP1448583A4 (fr) 2001-07-20 2002-07-19 Nouveau recepteur couple aux proteines g, gave8
CA002454427A CA2454427A1 (fr) 2001-07-20 2002-07-19 Nouveau recepteur couple aux proteines g, gave8
MXPA04000437A MXPA04000437A (es) 2001-07-20 2002-07-19 Un nuevo receptor acoplado a proteina g, gave8.
BR0211338-4A BR0211338A (pt) 2001-07-20 2002-07-19 Receptor acoplado à proteìna g, gave8
JP2003514877A JP2005522184A (ja) 2001-07-20 2002-07-19 新規のｇタンパク質共役受容体ｇａｖｅ８
IL15993902A IL159939A0 (en) 2001-07-20 2002-07-19 A novel g protein-coupled receptor, gave8
KR10-2004-7001067A KR20040017831A (ko) 2001-07-20 2002-07-19 신규한 ｇ 단백질 커플링된 수용체, ｇａｖｅ8
NO20040252A NO20040252L (no) 2001-07-20 2004-01-19 En ny G-proteinkoblet reseptor, GAVE8

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US30643401P 2001-07-20 2001-07-20
US60/306,434 2001-07-20
GB0125704.7 2001-10-26
GBGB0125704.7A GB0125704D0 (en) 2001-07-20 2001-10-26 A novel g protein-coupled receptor gaves

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WO2001036473A2 (fr) * 1999-11-16 2001-05-25 Pharmacia & Upjohn Company Recepteurs couples a une proteine g
EP1278850B1 (fr) * 2000-04-26 2007-06-13 Sanofi-Aventis Deutschland GmbH Recepteur edg8, sa preparation et son utilisation
AU2001276837A1 (en) * 2000-06-23 2002-01-08 Tularik, Inc. Human and mouse g-protein couipled receptors

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EP1448583A4 (fr) 2006-02-08
WO2003008560A8 (fr) 2004-06-24
TWI250209B (en) 2006-03-01
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