EP1173457A1

EP1173457A1 - Polynucleotide and polypeptide sequences encoding human organic anion transporter 6(hoatp6) and screening methods thereof

Info

Publication number: EP1173457A1
Application number: EP00910361A
Authority: EP
Inventors: Harma Ellens; John Field; Lin Yue
Original assignee: SmithKline Beecham Corp
Current assignee: SmithKline Beecham Corp
Priority date: 1999-03-04
Filing date: 2000-02-28
Publication date: 2002-01-23
Also published as: WO2000052024A1; EP1173457A4

Abstract

OATP6 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing OATP6 polypeptides and polynucleotides in therapy, and diagnostic assays for such. Futher disclosed is a method for performing a selection screen, whereby compounds are identified that neither agonize nor antagonize the activity of OATP6.

Description

POLYNUCLEOTIDE AND POLYPEPTTDE SEQUENCES ENCODING HUMAN ORGANIC ANION TRANSPORTER 6 (hOATPό) AND SCREENING METHODS THEREOF

Field of the Invention This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists, antagonists and/or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides.

Background of the Invention

The drug discovery process is currently undergoing a fundamental revolution as it embraces 'functional genomics', that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on 'positional cloning'. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.

Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterise further genes and their related polypeptides/proteins, as targets for drug discovery. Multi-specific drug transporters are present in cells having a barrier function, such as intestinal epithelial and brain microvessel endothelial cells. Other tissues, for example, liver and kidney, also contain multi-specific transporters that can mediate the excretion of drugs and metabolites. Information gained from using multi-specific transporters such as the human OATP6 gene product in cell based, membrane based, binding or other assays could enhance drug formulation, selection of formulation excipients, and compound design.

Summary of the Invention

The present invention relates to OATP6, in particular OATP6 polypeptides and OATP6 polynucleotides, recombinant materials and methods for their production. Furthermore, the present invention relates to a variant of human OATP6, the cDNA and amino acid sequences of which are set forth in SEQ ID NOs:3 and 4, respectively. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of cancer, inflammation,cardiovascular disease, central nervous system disorders, auto-immune disease, kidney and liver disease, or in identifying individuals who may handle drugs differently than "normal" individuals, hereinafter referred to as "the Diseases", amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with OATP6 imbalance with the identified compounds.

Another embodiment of this invention provides for methods to identify compounds that neither agonize nor antagonize OATP6. This invention further relates to the generation of in vitro and in vivo comparison data to predict oral absorption and pharmacokmetics in man. Such a comparison of data will enable selection of drugs with optimal pharmacokmetics in man, i e., good oral bioavailabihty, bram penetration, plasma half life, and minimum drug interaction. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with mappropnate OATP6 activity or levels.

Description of the Invention In a first aspect, the present invention relates to OATP6 polypeptides. Such peptides include isolated polypeptides comprising an ammo acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 or SEQ ID NO;4 over the entire length of SEQ ID NO:2 or SEQ ID NO:4. Such polypeptides include those comprising the ammo acid of SEQ ID NO:2 or SEQ ID NO:4 .

Further peptides of the present invention include isolated polypeptides in which the ammo acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NO:2 or SEQ ID NO:4 over the entire length of SEQ ID NO:2 or SEQ ID NO:4. Such polypeptides include the polypeptides of SEQ ID NO:2 and SEQ ID NO:4.

Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1 or in SEQ ID NO:3.

Polypeptides of the present invention are believed to be members of the organic amon transporter family of polypeptides. They are therefore of interest because they can be used to establish assays to predict oral absorbtion and pharmacokmetics and thus enhance compound and formulation design. These properties are hereinafter referred to as "OATP6 activity" or "OATP6 polypeptide activity" or "biological activity of OATP6". Also included amongst these activities are antigenic and lmmunogenic activities of said OATP6 polypeptides, in particular the antigenic and lmmunogenic activities of the polypeptide of SEQ ID NO:2. Preferably, a polypeptide of the present invention exhibits at least one biological activity of OATP6

The polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in puπfication such as multiple histidme residues, or an additional sequence for stability duπng recombmant production.

The present invention also includes include vaπants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteπstics. Typical such substitutions are among Ala, Val, Leu and

Be; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr. Particularly preferred are vaπants in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination. Polypeptides of the present invention can be prepared m any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for prepaπng such polypeptides are well understood in the art

In a further aspect, the present invention relates to OATP6 polynucleotides Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the ammo acid sequence of SEQ LD NO:2 or SEQ ID NO:4, over the entire length of SEQ ID NO.2 or SEQ ID NO:4. In this regard, polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide compπsing the nucleotide sequence contained m SEQ ID NO:l or SEQ ID NO:3 encoding the polypeptide of SEQ ID NO:2.

Further polynucleotides of the present invention include isolated polynucleotides compπsing a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2 or SEQ ID NO:4, over the entire coding region. In this regard, polynucleotides which have at least 97% identity are highly preferred, while those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO: 1 or SEQ ID NO:3 over the entire length of SEQ ID NO: 1 or SEQ ID NO:3. In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identify are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide compπsing the polynucleotide of SEQ ID NO: 1 or SEQ ID NO:3 as well as the polynucleotide of SEQ LD NO: 1 or SEQ ID NO:3

The invention also provides polynucleotides which are complementary to all the above described polynucleotides .

The nucleotide sequences of SEQ LD NO: 1 and SEQ LD NO:3 shows homology with rat LTSP (Genbank L27651). The nucleotide sequence of SEQ ID NO.1 is a cDNA sequence and compπses a polypeptide encoding sequence (nucleotide 15 to 1658) encoding a polypeptide of 548 ammo acids, the polypeptide of SEQ ID NO:2. The nucleotide sequence of SEQ ID NO: 3 is a cDNA sequence and compπses a polypeptide encoding sequence (nucleotide 15 to 1667) encoding a polypeptide of 551 ammo acids, the polypeptide of SEQ ID NO:4. The nucleotide sequence encoding the polypeptide of SEQ LD NO:2 or SEQ ID NO:4 may be identical to the polypeptide encoding sequence contained in SEQ LD NO: 1 or SEQ ID NO:3, respectively, or it may be a sequence other than the one contained in SEQ LD NO: 1 or SEQ LD NO:3, respectively, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ LD NO:2 or SEQ LD NO:4. The polypeptides set forth in SEQ ID NO:2 and SEQ ID NO:4 are structurally related to other proteins of the organic anion transporters family, having homology and/or structural similanty with rat LSTP (G.D Simonson, et al , J Cell Sci. 107, 1065-1072, 1994; T. Sekme et al. FEBS Lett . Jun 12;429(2): 179-82, 1998). Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one OATP6 activity

Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library deπved from mRNA in cells of human kidney and liver, using the expressed sequence tag (EST) analysis (Adams, M.D., et al Science (1991) 252: 1651-1656; Adams, M.D. et al , Nature, (1992) 355:632-634; Adams, M.D., et al , Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbraπes or can be synthesized using well known and commercially available techniques. When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide m reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates puπfication of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and descπbed in Gentz et al , Proc NatlAcad Set USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-codmg 5' and 3' sequences, such as transcπbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mPvNA.

Further embodiments of the present invention include polynucleotides encoding polypeptide vaπants which compπse the ammo acid sequence of SEQ LD NO.2 or SEQ ID NO:4 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, ammo acid residues are substituted, deleted or added, in any combination.

Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO:l or m SEQ ID NO: 3, may be used as hybπdization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similaπty to SEQ LD NO: 1 or to SEQ ID NO:3. Typically these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent. The probes or pπmers will generally compπse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.

A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than human, may be obtained by a process which compπses the steps of screening an appropπate library under stringent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof or SEQ ID NO: 3 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybπdization techniques are well known to the skilled artisan Preferred stringent hybπdization conditions include overnight incubation at 42°C in a solution compπsing: 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 M sodium phosphate (pH 7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters m O.lx SSC at about 65°C. Thus the present invention also includes polynucleotides obtainable by screenmg an appropπate library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO:l or a fragment thereof or SEQ LD NO:3 or a fragment thereof. The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, m that the region coding for the polypeptide is cut short at the 5' end of the cDNA. This is a consequence of reverse transcπptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the rnRNA template during 1st strand cDNA synthesis. There are several methods available and well known to those skilled in the art to obtain full- length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al, PNAS USA 85, 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon™' technology (Clontech Laboratoπes Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from rnRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide primers. The PCR reaction is then repeated using 'nested' primers, that is, pπmers designed to anneal withm the amplified product (typically an adaptor specific pπmer that anneals further 3' in the adaptor sequence and a gene specific pπmer that anneals further 5' in the known gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer. Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells compπsing expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which compπse a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the present invention

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods descπbed m many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook, et al, Molecular Clonmg: A Laboratory Manual, 2nd Ed., Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, N.Y. (1989). Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropπate hosts include bacteπal cells, such as streptococci, staphylococci, E coli, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. A great vaπety of expression systems can be used, for instance, chromosomal, episomal and virus-denved systems, e g , vectors deπved from bacteπal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropπate nucleotide sequence may be mserted into an expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al, MOLECULAR CLONING, A LABORA TORY MANUAL (supra).

Appropπate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the peπplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. If a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested pπor to use m the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered m order to recover and purify the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered. Polypeptides of the present invention can be recovered and puπfied from recombmant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for puπfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or puπfication.

This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characteπzed by the polynucleotide of SEQ LD NO:l or SEQ ID NO: 3 that is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a vaπety of techniques. A diagnostic reagent for the gene may also be used to determine appropπate drug dosing regimes. Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, uπne, saliva, tissue biopsy or autopsy material The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques pπor to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product m compaπson to the normal genotype. Point mutations can be identified by hybπdizmg amplified DNA to labeled OATP6 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denatuπng agents, or by direct DNA sequencing (eg., Myers, et al , Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton, etal, Proc Natl Acad Sci USA (1985) 85: 4397-4401). In another embodiment, an array of o gonucleotides probes compπsing OATP6 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, e g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a vaπety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example:

M. Chee, et al , Science, Vol 274, pp 610-613 (1996))

The diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the OATP6 gene by the methods descπbed In addition, such diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or rnRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample deπved from a host are well-known to those of skill m the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

Thus m another aspect, the present invention relates to a diagnostic kit which compπses:

(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO. 1, or a fragment thereof or SEQ ID NO: 3 or a fragment thereof ;

(b) a nucleotide sequence complementary to that of (a),

(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2 or a fragment thereof or SEQ ID NO:4 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2 or SEQ ID NO:3.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly cancer, ιnflammatιon,cardιovascular disease, central nervous system disorders, autoimmune disease, kidney and liver disease , amongst others, or in identifying individuals who may handle drugs differently than "normal" individuals.

The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to, and can hybπdize with, a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. 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 in, for example, V. McKusick, Mende an Inheπtance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (cornheπtance of physically adjacent genes)

The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not m any normal individuals, then the mutation is likely to be the causative agent of the disease.

The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them, can also be used as lmmunogens to produce antibodies lmmunospecific for polypeptides of the present invention. The term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides m the pπor art.

Antibodies generated against polypeptides of the present invention may be obtained by admimsteπng the polypeptides or epitope-beaπng fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybπdoma technique (Kohler, G. and Milstem, C, Nature (1975) 256:495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al , Immunology Today (1983) 4:72) and the EBV- hybπdoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp

77-96, Alan R. Liss, Inc., 1985)

Techniques for the production of single chain antibodies, such as those descπbed in U.S. Patent No. 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies .

The above-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to puπfy the polypeptides by affinity chromatography

Antibodies against polypeptides of the present invention may also be employed to treat the Diseases, amongst others. In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of lmmunoglobulms of various subclasses (IgG, IgM, IgA, IgE). Preferred as an lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914

Another aspect of the invention relates to a method for inducing an lmmunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.

A further aspect of the invention relates to an lmmunological/vaccme formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention. The vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection) Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requiπng only the addition of the sterile liquid carrier immediately pnor to use. The vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-m water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expeπmentation.

Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide. Accordingly, a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned. Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures. Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology l(2):Chapter 5 (1991)).

The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beaπng the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active polypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring OATP6 activity in the mixture, and comparing the OATP6 activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and OATP6 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al, J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995))

The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of rnRNA and polypeptide m cells. For example, an ELISA assay may be constructed for measuπng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

The polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslmkmg assays which the polypeptide is labeled with a radioactive isotope

(for instance, ^I), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and specfroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art

Examples of potential polypeptide antagonists include antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the present invention; (c) a cell membrane expressing a polypeptide of the present invention; or

(d) antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2 or SEQ ID NO:4.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. It will be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by

(a) determining in the first instance the three-dimensional structure of the polypeptide;

(b) deducing the three-dimensional structure for the likely reactive or binding sιte(s) of an agonist, antagonist or inhibitor,

(c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors.

It will be further appreciated that this will normally be an interactive process Organic anion transporters, such as OATP6, are present in cells having a barπer function, such as intestinal epithelial cells, brain microvessel endothehal cells, kidney epithelial cells, and liver hepatocytes. It was recently recognized that these transporters contribute to poor intestinal absorption, poor penetration into the bram, rapid plasma clearance and vaπabihty, as well as drug interactions. In a preferred embodiment, the present invention relates to the use of OATP6 polypeptides, polynucleotides, and recombinant materials thereof m selection screens to identify compounds that are neither agonists nor antagonist/inhibitors of OATP6. The data from such a selection screen is expected to provide in vitro and in vivo comparisons to predict oral absorption and pharmacokmetics m humans The ability to make such a compaπson of data will enhance drug formulation design through the identification of compounds with optimal development characteπstics, i.e., high oral bioavailabihty, ULD (once a day) dosmg, reduced drug interactions, reduced variability, and reduced food effects, specifically to avoid interacting with OATP6.

In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, cancer, ιnflammatιon,cardιovascular disease, central nervous system disorders, auto-immune disease, kidney and liver disease , amongst others, or in identifying individuals who may handle drugs differently than "normal" individuals, , related to either an excess of, or an under-expression of, OATP6 polypeptide activity.

If the activity of the polypeptide is in excess, several approaches are available. One approach compπses administering to a subject in need thereof an inhibitor compound (antagonist) as hereinabove descπbed, optionally in combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide may be administered. Typical examples of such competitors include fragments of the OATP6 polypeptide

In still another approach, expression of the gene encoding endogenous OATP6 polypeptide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene

Expression, CRC Press, Boca Raton, FL (1988)). Alternatively, ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee, et al, Nucleic Acids Res (1979) 6:3073; Cooney, et al , Science (1988) 241:456; Dervan, et al , Science (1991) 251: 1360). These ohgomers can be administered er se or the relevant oligomers can be expressed in vivo. For treating abnormal conditions related to an under-expression of OATP6 and its activity, several approaches are also available. One approach compnses admmisteπng to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present invention, i.e , an agonist as descnbed above, in combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of OATP6 by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression m a replication defective retro viral vector, as discussed above The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engmeenng cells in vivo and expression of the polypeptide in vivo. For an overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) m Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical earner.

In a further aspect, the present invention provides for pharmaceutical compositions compπsing a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient. Such earners include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or mtrapeπtoneal, can be used Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteπc or an encapsulated formulation, oral administration may also be possible Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.

The dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide vanations in the needed dosage, however, are to be expected in view of the vanety of compounds available and the diffeπng efficiencies of vanous routes of administration. For example, oral administration would be expected to require higher dosages than administration by mtravenous injection. Vanations in these dosage levels can be adjusted using standard empiπcal routines for optimization, as is well understood in the art

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as descπbed above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.

Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology. This is most easily facilitated by stoπng the sequence m a computer readable medium and then using the stored data to search a sequence database using well known searching tools, such as GCC. Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1, SEQ ID NO:3 and/or a polypeptide sequence encoded thereby.

The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulm expression library. "Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs m nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein "Polynucleotide" generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition,

"polynucleotide" refers to tπple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases mclude, for example, tntylated bases and unusual bases such as mosine. A vanety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly refened to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids. "Polypeptides" include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known m the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitmation (see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 m POSTTRANSLATIONAL COVALENT

MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al , "Analysis for protein modifications and nonprotem cofactors", Meth Enzymol (1990) 182:626- 646 and Rattan et al , "Protein Synthesis: Post-translational Modifications and Agmg", Ann NYAcad "Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non- naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M

Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SLAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Prefeπed computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NTH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity. Preferred parameters for polypeptide sequence comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992) Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps). Preferred parameters for polynucleotide comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol 48 443-453 (1970)

Comparison matrix: matches = +10, mismatch = 0

Gap Penalty: 50

Gap Length Penalty. 3 Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ED NO: 1 or SEQ ED NO:3, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups withm the reference sequence The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ED NO: 1 or SEQ ED NO: 3 by the numerical percent of the respective percent ιdentιty(dιvιded by 100) and subtracting that product from said total number of nucleotides in SEQ LD NO: 1 or SEQ ED NO:3, or

ⁿn ^{≤ x}n ^" (^xn ^• y)> wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ LD NO: 1 or SEQ ED NO:3, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for

90%, 0.95 for 95%, etc., and wherein any non-mteger product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ED NO:2 or SEQ LD N0 4 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations. Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ED NO:2 or SEQ ID NO:4, that is be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids m SEQ ED NO:2 or SEQ ED NO:4 by the numerical percent of the respective percent ιdentιty(dιvιded by 100) and then subtracting that product from said total number of ammo acids in SEQ ED NO:2 or SEQ ID NO:4, or: n_a<x_a - (x_a • y),

wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo acids in SEQ ED NO:2 or SEQ ED NO:4, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc., and wherein any non-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 discloses fusion proteins comprising vanous portions of constant region of immunoglobulm molecules together with another human protein or part thereof. In many cases, employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e g , EP-A 0232 262], On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified.

All publications, including but not limited to patents and patent applications, cited m this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth

Claims

What is claimed is:

1. An isolated polypeptide selected from the group consisting of

(I) an isolated polypeptide comprising an ammo acid sequence selected from the group having at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity; to the ammo acid sequence of SEQ ID NO:2 over the entire length of SEQ ED

NO:2;

(n) an isolated polypeptide comprising the ammo acid sequence of SEQ ED NO:2; or

(in) an isolated polypeptide which is the ammo acid sequence of SEQ ED NO:2.

2. An isolated polynucleotide selected from the group consisting of

(I) an isolated polynucleotide compnsmg a nucleotide sequence encoding a polypeptide that has at least

(a) 70% identity;

(b) 80% identity; (c) 90% identity; or

(d) 95% identity; to the ammo acid sequence of SEQ ED NO:2, over the entire length of SEQ ED NO:2; (n) an isolated polynucleotide compπsing a nucleotide sequence that has at least:

(a) 70% identity; (b) 80% identity;

(c) 90% identity; or

(d) 95% identity, over its entire length to a nucleotide sequence encoding the polypeptide of SEQ ED NO:2;

(in) an isolated polynucleotide compnsmg a nucleotide sequence which has at least.

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity,

to that of SEQ ED NO: 1 over the entire length of SEQ ED NO: 1 ; (iv) an isolated polynucleotide compnsmg a nucleotide sequence encoding the polypeptide of

SEQ ED NO:2;

(v) an isolated polynucleotide which is the polynucleotide of SEQ LD NO: 1; or

(vi) an isolated polynucleotide obtainable by screenmg an appropnate library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof. ; or a nucleotide sequence complementary to said isolated polynucleotide.

3. An antibody lmmunospecific for the polypeptide of claim 1

4. A method for the treatment of a subject:

(l) in need of enhanced activity or expression of the polypeptide of claim 1 comprising:

(a) admmistenng to the subject a therapeutically effective amount of an agonist to said polypeptide; and/or

(b) providing to the subject an isolated polynucleotide compnsmg a nucleotide sequence encoding said polypeptide in a form so as to effect production of said polypeptide activity in vivo.; or

(n) having need to inhibit activity or expression of the polypeptide of claim 1 comprising: (a) admmistenng to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or

(b) admmistenng to the subject a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding said polypeptide; and/or (c) admmistenng to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its hgand, substrate , or receptor.

5. A process for diagnosing a disease or a susceptibility to a disease m a subject related to expression or activity of the polypeptide of claim n a subject comprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide m the genome of said subject; and/or

(b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said subject.

6. A method for screening to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which compπses a method selected from the group consisting of:

(a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;

(b) measunng the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor;

(c) testing whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide;

(d) mixing a candidate compound with a solution containing a polypeptide of claim 1 , to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard; or (e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide m cells, using for instance, an ELISA assay

7. An agonist or an antagonist of the polypeptide of claim 1.

8. An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell.

9. A process for producing a recombinant host cell comprising transforming or transfectmg a cell with the expression system of claim 8 such that the host cell, under appropnate culture conditions, produces a polypeptide compnsmg an ammo acid sequence having at least 70% identity to the ammo acid sequence of SEQ ED NO:2 over the entire length of SEQ ED NO:2.

10. A recombinant host cell produced by the process of claim 9.

11. A membrane of a recombinant host cell of claim 10 expressing a polypeptide comprising an ammo acid sequence having at least 70% identity to the ammo acid sequence of SEQ ED NO:2 over the entire length of SEQ LD NO:2

12. A process for producing a polypeptide comprising cultunng a host cell of claim 10 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.

13. A method for screening to identify compounds that neither agonize nor antagonize the activity of the polypeptide of Claim 1, compnsmg a method selected from the group consisting of:

(a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,

(b) measunng the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor; (c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide;

(d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measuπng activity of the polypeptide in the mixture, and companng the activity of the mixture to a standard; or

(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells.

14. An isolated polypeptide selected from the group consisting of:

(a) 70% identity;

(b) 80% identity; (c) 90% identity; or

(d) 95% identity; to the ammo acid sequence of SEQ ED NO:4 over the entire length of SEQ ED NO:4;

(n) an isolated polypeptide comprising the amino acid sequence of SEQ ED NO:4 or (in) an isolated polypeptide which is the ammo acid sequence of SEQ ED NO:4.

15. An isolated polynucleotide selected from the group consisting of:

(l) an isolated polynucleotide compnsmg a nucleotide sequence encoding a polypeptide that has at least (a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity; to the ammo acid sequence of SEQ ED NO.4, over the entire length of SEQ ED NO:4; (n) an isolated polynucleotide compnsmg a nucleotide sequence that has at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or (d) 95% identity; over its entire length to a nucleotide sequence encoding the polypeptide of SEQ LD NO:4;

(in) an isolated polynucleotide compnsmg a nucleotide sequence which has at least:

(a) 70% identity; (b) 80% identity;

(c) 90% identity; or

(d) 95% identity; to that of SEQ ED NO:3 over the entire length of SEQ ED NO:3;

(iv) an isolated polynucleotide compnsmg a nucleotide sequence encoding the polypeptide of SEQ ED NO:4;

(v) an isolated polynucleotide which is the polynucleotide of SEQ ED NO: 3; or

(vi) an isolated polynucleotide obtainable by screening an appropnate library under stringent hybπdization conditions with a labeled probe having the sequence of SEQ ED NO: 3 or a fragment thereof; or a nucleotide sequence complementary to said isolated polynucleotide.

16. An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim 14 when said expression system is present in a compatible host cell.

17. A process for producing a recombinant host cell comprising transforming or fransfectmg a cell with the expression system of claim 16 such that the host cell, under appropnate culture conditions, produces a polypeptide comprising an ammo acid sequence having at least 70% identity to the ammo acid sequence of SEQ ID NO-4 over the entire length of SEQ ED NO:4.

18. A recombinant host cell produced by the process of claim 17.

19. A membrane of a recombinant host cell of claim 18 expressing a polypeptide comprising an ammo acid sequence having at least 70% identity to the ammo acid sequence of SEQ ED NO:4 over the entire length of SEQ ED NO:4.

20. A process for producing a polypeptide comprising culturmg a host cell of claim 19 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.

21. A method for screening to identify compounds that neither agonize nor antagonize the activity of the polypeptide of claim 14, compnsmg a method selected from the group consisting of:

(b) measuπng the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor;

(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide;

(d) mixing a candidate compound with a solution containing a polypeptide of claim 14, to form a mixture, measuπng activity of the polypeptide in the mixture, and compaπng the activity of the mixture to a standard; or (e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells.