WO2001009166A1 - Utilisation d'octoray en tant que recepteur couple a la proteine g - Google Patents

Utilisation d'octoray en tant que recepteur couple a la proteine g Download PDF

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Publication number
WO2001009166A1
WO2001009166A1 PCT/US2000/020005 US0020005W WO0109166A1 WO 2001009166 A1 WO2001009166 A1 WO 2001009166A1 US 0020005 W US0020005 W US 0020005W WO 0109166 A1 WO0109166 A1 WO 0109166A1
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polypeptide
polynucleotide
sequence
seq
isolated
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PCT/US2000/020005
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English (en)
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Derk J. Bergsma
Nabil A. Elshourbagy
Stephanie F. Guerrera
Nandu Gattu
Jeffrey L. Mooney
Lisa Vawter
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Smithkline Beecham Corporation
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Publication of WO2001009166A1 publication Critical patent/WO2001009166A1/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
    • 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

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use m diagnosis and identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
  • proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991 , 351:353-354).
  • these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins.
  • Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamme (Kobilka, B.K., et al., Proc Natl Acad.
  • G-protems themselves, effector proteins, e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kmase A and protein kmase C (Simon, M I., et al., Science, 1991, 252:802-8)
  • effector proteins e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase
  • actuator proteins e.g., protein kmase A and protein kmase C (Simon, M I., et al., Science, 1991, 252:802-8)
  • the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell.
  • GTP also influences hormone binding.
  • a G-protem connects the hormone receptor to adenylate cyclase G- protein was shown to exchange GTP for bound GDP when activated by a hormone receptor The GTP- carrymg form then binds to activated adenylate cyclase Hydrolysis of GTP to GDP, catalyzed by the G-protem itself, returns the G-protem to its basal, inactive form.
  • the G-protem serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal
  • G-protem coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
  • G-protem coupled receptors (otherwise known as 7TM receptors) have been charactenzed as including these seven conserved hydrophobic stretches of about 20 to 30 ammo acids, connecting at least eight divergent hydrophilic loops.
  • the G-protem family of coupled receptors includes dopamine receptors which bmd to neuroleptic drugs used for treating psychotic and neurological disorders Other examples of members of this family include, but are not limited to, calcitonm, adrenergic, endothelm, cAMP, adenosme, muscannic, acetylchohne, serotonin, histamine, thromb , kmin, follicle stimulating hormone, opsins, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors
  • G-protem coupled receptors have single conserved cysteme residues m each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure.
  • the 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7.
  • TM3 has been implicated m signal transduction.
  • Phosphorylation and hpidation (palmitylation or farnesylation) of cysteme residues can influence signal transduction of some G-protem coupled receptors.
  • Most G-protem coupled receptors contain potential phosphorylation sites within the third cytoplasrmc loop and/or the carboxy terminus.
  • G-protem coupled receptors such as the ⁇ - adrenoreceptor
  • phosphorylation by protein kmase A and/or specific receptor kinases mediates receptor desensitization.
  • the ligand binding sites of G-protem coupled receptors are believed to compnse hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protem coupled receptors
  • the hydrophilic side of each G-protem coupled receptor transmembrane helix is postulated to face inward and form a polar ligand binding site.
  • TM3 has been implicated in several G-protem coupled receptors as having a ligand bmdmg site, such as the TM3 aspartate residue TM5 sennes, a TM6 asparagine and
  • TM6 or TM7 phenylalanmes or tyrosmes are also implicated in ligand binding
  • G-protem coupled receptors can be lntracellularly coupled by heterot ⁇ menc G-protems to va ⁇ ous mtracellular enzymes, ion channels and transporters (see, Johnson et al , Endoc Rev , 1989, 10:317-331). Different G-protem ⁇ -subumts preferentially stimulate particular effectors to modulate va ⁇ ous biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protem coupled receptors has been identified as an important mechanism for the regulation of G-protem couplmg of some G-protem coupled receptors. G-protem coupled receptors are found m numerous sites within a mammalian host.
  • the present invention relates to Octoray, in particular Octoray polypeptides and Octoray polynucleotides, recombmant mate ⁇ als and methods for their production.
  • Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pecto ⁇ s, myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, deh ⁇ um, dementia, and severe mental retardation; and dyskmesias, such as Huntmgton's disease or Gilles dela Tourett's syndrome
  • the invention relates to diagnostic assays for detecting diseases associated with mapprop ⁇ ate Octoray activity or levels.
  • the present invention relates to Octoray polypeptides.
  • Such polypeptides include:
  • polypeptide sequence of SEQ ID NO:2 (e) the polypeptide sequence of SEQ ID NO:2; and (f) an isolated polypeptide having or comp ⁇ smg a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2;
  • Polypeptides of the present invention are believed to be members of the 7-TM (G-Protem Coupled) family of polypeptides. They are therefore of interest because 7-TM receptors are proven to be an important therapeutic target for drug discovery.
  • a polypeptide of the present invention exhibits at least one biological activity of Octoray.
  • Polypeptides of the present invention also mcludes va ⁇ ants of the aforementioned polypeptides, including all allehc forms and splice va ⁇ ants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof.
  • Particularly preferred vanants are those in which several, for mstance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, m any combination.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide comp ⁇ smg an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO: 2.
  • Preferred fragments are biologically active fragments that mediate the biological activity of Octoray, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or lmmunogenic in an animal, especially in a human.
  • Fragments of the polypeptides of the invention may be employed for producing the co ⁇ espondmg full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • 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 precursor or a fusion protein. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production.
  • Polypeptides of the present invention can be prepared in any suitable manner, for mstance by isolation form naturally occurring sources, from genetically engineered host cells comp ⁇ smg expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.
  • the present invention relates to Octoray polynucleotides.
  • Such polynucleotides include- (a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95%, 96%, 97%,
  • an isolated polynucleotide comp ⁇ smg a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
  • polynucleotides that are fragments and va ⁇ ants of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof.
  • Preferred fragments of polynucleotides of the present mvention include an isolated polynucleotide comprising an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO 1 , or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ED NO.
  • va ⁇ ants of polynucleotides of the present invention include splice va ⁇ ants, allehc variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • Polynucleotides of the present mvention also include polynucleotides encodmg polypeptide va ⁇ ants that comp ⁇ se the ammo acid sequence of SEQ ID NO:2 and m which several, for mstance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination.
  • the present mvention provides polynucleotides that are RNA transc ⁇ pts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that:
  • (a) comprises an RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
  • (b) is the RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
  • (c) comprises an RNA transc ⁇ pt of the DNA sequence of SEQ ID NO: 1 ; or (d) is the RNA transc ⁇ pt of the DNA sequence of SEQ ID NO: 1 ; and RNA polynucleotides that are complementary thereto.
  • the polynucleotide sequence of SEQ ID NO:l shows homology with KIAAOOOl (Nomura N., Miyajima N., Sazuka T, Tanaka A, Kawarabayashi Y, Sato S, Nagase T, Seki N, Ishikawa K, Tabata S 1994. DNA Res. 1, 27-35).
  • the polynucleotide sequence of SEQ ID NO:l is a cDNA sequence that encodes the polypeptide of SEQ ED NO:2.
  • SEQ ID NO:2 may be identical to the polypeptide encodmg sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ED NO: 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polypeptide of the SEQ ID NO:2 is related to other protems of the 7-TM family, having homology and/or structural sirmla ⁇ ty with KIAAOOOl (Normura N, Miyajima N, Sazuka T, Tanaka A, Kawarabayashi Y, Sato S, Nagase T, Seki
  • 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 Octoray activity
  • Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library de ⁇ ved from mRNA in cells of human placenta and testis, (see for mstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbra ⁇ es or can be synthesized using well known and commercially available techniques.
  • 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- prote sequence, or other fusion peptide portions.
  • a marker sequence that facilitates pu ⁇ fication of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and desc ⁇ bed in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome bmdmg sites and sequences that stabilize mRNA.
  • Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO: 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers for a nucleic acid amplification reaction (for instance, PCR). Such probes and p ⁇ mers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence simila ⁇ ty to SEQ ID NO.1 , typically at least 95% identity.
  • Preferred probes and p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
  • a polynucleotide encoding a polypeptide of the present invention, mcludmg homologs from species other than human, may be obtained by a process comp ⁇ smg the steps of screening a library under st ⁇ ngent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ID NO" 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • hyb ⁇ dization techniques are well known to the skilled artisan.
  • Preferred st ⁇ ngent hyb ⁇ dization conditions include overnight incubation at 42°C in a solution comp ⁇ smg: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM 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 0 lx SSC at about 65°C.
  • a solution comp ⁇ smg: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM 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 0 lx SSC at about 65°C.
  • the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screenmg a library under st ⁇ ngent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides.
  • PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal withm the amplified product (typically an adaptor specific p ⁇ mer that anneals further 3' m the adaptor sequence and a gene specific p ⁇ mer that anneals further 5' in the known gene sequence).
  • primers designed to anneal withm the amplified product typically an adaptor specific p ⁇ mer that anneals further 3' m the adaptor sequence and a gene specific p ⁇ mer 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' p ⁇ mer.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comp ⁇ smg expression systems.
  • the present invention relates to expression systems comp ⁇ smg 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 de ⁇ ved from the DNA constructs of the present mvention
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Polynucleotides may be introduced into host cells by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al.(ibid).
  • Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • Representative examples of appropriate hosts include bacterial cells, such as Streptococci,
  • Staphylococci E. coli, Streptomyces and Bacillus subtilis 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
  • plant cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells.
  • expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g. , vectors derived from bacterial plasmids, from bacteriophage, 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 derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
  • the appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, (ibid).
  • Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • 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 prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification. Polynucleotides of the present mvention may be used as diagnostic reagents, through detecting mutations in the associated gene.
  • Detection of a mutated form of the gene characte ⁇ zed by the polynucleotide of SEQ ID NO.1 in the cDNA or genomic sequence and which 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 spatial or temporal expression of the gene.
  • Individuals carrying mutations m the gene may be detected at the DNA level by a va ⁇ ety of techniques well known in the art.
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mate ⁇ al.
  • the genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques pnor 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 in compa ⁇ son to the normal genotype. Pomt mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled Octoray nucleotide sequences.
  • DNA sequence difference may also be detected by alterations m the electrophoretic mobility of DNA fragments in gels, with or without denatu ⁇ ng agents, or by direct DNA sequencing (see, for instance, 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 et al , Proc Natl Acad Sci USA (1985) 85: 4397-4401).
  • An array of oligonucleotides probes comp ⁇ smg Octoray polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations.
  • Such arrays are preferably high density arrays or g ⁇ ds
  • Array technology methods are well known and have general applicability and can be used to address a va ⁇ ety of questions in molecular genetics including gene expression, genetic linkage, and genetic va ⁇ abihty, see, for example, M.Chee et al., Science, 274, 610- 613 (1996) and other references cited therein.
  • Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention 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 mstance 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 de ⁇ ved 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
  • the present invention relates to a diagnostic kit comprising:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transcript thereof;
  • kits may comprise a substantial component.
  • Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others
  • the polynucleotide sequences of the present mvention are valuable for chromosome localisation studies.
  • the sequence is specifically targeted to, and can hyb ⁇ dize 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 m 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, Mendehan Inhe ⁇ tance in Man (available on-lme through Johns Hopkins University Welch Medical Library).
  • 93 PCRs are performed using primers designed from the gene of interest on RH DNAs Each of these DNAs contains random human genomic fragments maintained in a hamster background (human / hamster hybrid cell lines) These PCRs result in 93 scores indicating the presence or absence of the PCR product of the gene of interest These scores are compared with scores created using PCR products from genomic sequences of known location. This compa ⁇ son is conducted at http://www.genome.wi.mit.edu/.
  • the polynucleotide sequences of the present mvention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides m tissues, by detecting the mRNAs that encode them.
  • the techniques used are well known in the art and include m situ hyd ⁇ disation techniques to clones arrayed on a g ⁇ d, such as cDNA microarray hyb ⁇ dization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR.
  • a preferred method uses the TAQMAN (Trade mark) technology available from Perk Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropnate expression thereof in disease.
  • mapprop ⁇ ate expression may be of a temporal, spatial or simply quantitative nature.
  • a further aspect of the present mvention relates to antibodies.
  • the polypeptides of the invention or their fragments, or cells expressmg them, can be used as lmmunogens to produce antibodies that are 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 the pnor art.
  • Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, or cells to an animal, preferably a non- human animal, using routine protocols.
  • an animal preferably a non- human animal
  • any techmque which provides antibodies produced by continuous cell line cultures can be used. Examples include the hyb ⁇ doma technique (Kohler, G.
  • No 4,946,778 can also be adapted to produce single chain antibodies to polypeptides of this invention.
  • transgemc mice, or other organisms, including other mammals may be used to express humanized antibodies
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to punfy the polypeptides by affinity chromatography.
  • Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others.
  • polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an lmmunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established withm the individual or not.
  • a method for inducing an lmmunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established withm the individual or not.
  • An lmmunological response m a mammal may also be induced by a method comp ⁇ ses dehve ⁇ ng 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 lmmunological response to produce antibody to protect said animal from diseases of the invention.
  • One way of admmiste ⁇ ng the vector is by accelerating it into the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comp ⁇ se DNA, RNA, a modified nucleic acid, or a DNA/RNA hyb ⁇ d.
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
  • the 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 mtradermal injection).
  • parenterally for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous ste ⁇ le injection solutions that may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes that render the formulation mstonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that 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-d ⁇ ed condition requi ⁇ ng only the addition of the ste ⁇ le liquid earner immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in 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 expe ⁇ mentation.
  • Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, m particular the diseases of the invention herembefore mentioned. It is therefore useful to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, m a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the mvention as hereinbefore mentioned. Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es, collections of chemical compounds, and natural product mixtures.
  • Such agonists or antagonists so-identified may be natural or modified substrates, gands, receptors, enzymes, etc., as the case may be, of the polypeptide, a structural or functional mimetic thereof (see Cohgan et al , Current Protocols m Immunology l(2):Chapter 5 (1991)) or a small molecule.
  • Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
  • the screening method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes bea ⁇ ng the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g agonist or antagonist). 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 approp ⁇ ate to the cells bea ⁇ ng 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. Further, the screening methods may simply comp ⁇ se the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a Octoray activity in the mixture, and compa ⁇ ng the Octoray activity of the mixture to a control mixture which contains no candidate compound.
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384- well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246,
  • Fusion proteins such as those made from Fc portion and Octoray polypeptide, as hereinbefore desc ⁇ bed, 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))
  • 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 mRNA and polypeptide in cells.
  • an ELISA assay may be constructed for measu ⁇ ng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known m the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • a polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor bmdmg techniques known the art. These include, but are not limited to, ligand binding and crosslmkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), 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 spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
  • antagonists of polypeptides of the present invention include antibodies or, in some cases, ohgonucleotides or proteins that are closely related to the hgands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g , a fragment of the hgands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • Transgenic technology may also involve the use of transgenic technology and Octoray gene.
  • the art of constructing transgenic animals is well established.
  • the Octoray gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts.
  • Particularly useful transgenic animals are so-called "knock-" animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-m transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target.
  • transgenic animals are so-called "knock-out" animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence m a cell is partially or completely annulled
  • the gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells m the animal.
  • Transgenic animal technology also offers a whole animal expression-clonmg system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
  • Screening kits for use m the above described methods form a further aspect of the present invention.
  • Such screening kits comprise:
  • polypeptide of the present invention (d) an antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an
  • Isolated means altered “by the hand of man” from its natural state, i e., if it occurs in nature, it has been changed or removed from its o ⁇ gmal environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism 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.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said organism, which organism may be living or non-living.
  • Polynucleotide generally refers to any poly ⁇ bonucleotide (RNA) or polydeox ⁇ bonucleotide (DNA), which may be unmodified 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 smgle-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • 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 include, for example, t ⁇ tylated bases and unusual bases such as inosme.
  • 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 characte ⁇ stic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
  • Polypeptide refers to any polypeptide comp ⁇ sing 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 referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally refe ⁇ ed 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 that are well known in the art. Such modifications are well desc ⁇ bed in basic texts and m 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.
  • 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- ⁇ bosylation, amidation, biotmylation, covalent attachment of flavin, 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 phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitmation (see, for
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. “Fragment” of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.
  • Va ⁇ ant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof.
  • a typical va ⁇ ant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the va ⁇ ant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical va ⁇ ant of a polypeptide differs m ammo acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the va ⁇ ant are closely similar overall and, m many regions, identical.
  • a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions m any combination.
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr.
  • a variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a va ⁇ ant that is not known to occur naturally
  • Non-naturally occur ⁇ ng va ⁇ ants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis
  • va ⁇ ants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ⁇ bosylation and the like.
  • Embodiments include methylation of the N-termmal ammo acid, phosphorylations of se ⁇ nes and threonmes and modification of C-terminal glycmes
  • Allele refers to one of two or more alternative forms of a gene occur ⁇ ng at a given locus in the genome
  • Polymorphism refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position m the genome withm a population
  • SNP Single Nucleotide Polymorphism
  • SNP Single Nucleotide Polymorphism
  • An SNP may occur withm a gene or withm mtergemc regions of the genome SNPs can be assayed using Allele Specific Amplification (ASA)
  • ASA Allele Specific Amplification
  • a common primer is used m reverse complement to the polymorphism being assayed. This common primer can be between 50 and 1500 bps from the polymorphic base.
  • the other two (or more) p ⁇ mers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism.
  • Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.
  • RNA molecules produced from RNA molecules initially transc ⁇ bed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
  • Alternative RNA splicing occurs when a primary RNA transc ⁇ pt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences.
  • the term splice variant also refers to the proteins encoded by the above cDNA molecules.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compa ⁇ ng the sequences. In general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.
  • % Identity For sequences where there is not an exact correspondence, a “% identity” may be determined
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment.
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
  • Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences.
  • similar ⁇ ty means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated "score” from which the "% similarity" of the two sequences can then be determined.
  • BESTFIT is more suited to compa ⁇ ng two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences, finding a "maximum simila ⁇ ty", according to the algo ⁇ thm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970).
  • GAP is more suited to compa ⁇ ng sequences that are approximately the same length and an alignment is expected over the entire length.
  • the parameters "Gap Weight” and "Length Weight” used m each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
  • % identities and simila ⁇ ties are determined when the two sequences being compared are optimally aligned
  • the BLOSUM62 ammo acid substitution matnx (Hemkoff S and Hemkoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into ammo acid sequences before comparison
  • the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore desc ⁇ bed
  • Identity Index is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence.
  • a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
  • Identity Index of 0 95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 ammo acids of the reference sequence. Such differences are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These differences may occur at the ammo- or carboxy- terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids m the reference sequence or in one or more contiguous groups withm the reference sequence.
  • an average of up to 5 m every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed.
  • n a is the number of nucleotide or ammo acid differences
  • x a is the total number of nucleotides or ammo acids in SEQ ID NO: 1 or SEQ ID NO.2, respectively,
  • I is the Identity Index , • is the symbol for the multiplication operator, and m which any non-mteger product of x a and I is rounded down to the nearest integer p ⁇ or to subtracting it from x a
  • “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence Such relatedness may be quantified by determining the degree of identity and/or simila ⁇ ty between the two sequences as hereinbefore defined Falling withm this gene ⁇ c term are the terms "ortholog", and “paralog” "Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species "Paralog” refers to a polynucleotide or polypeptide that within the same species which is functionally similar "Fusion protein” refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 533-A discloses fusion proteins comp ⁇ smg va ⁇ ous portions of constant region of immunoglobulin molecules together with another human protein or part thereof In many cases, employing

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Abstract

L'invention concerne des polypeptides et des polynucléotides Octoray, et des méthodes de production desdits polypeptides par des techniques recombinantes. Elle concerne en outre des méthodes d'utilisation des polypeptides et des polynucléotides Octoray à des fins de diagnostic.
PCT/US2000/020005 1999-07-29 2000-07-21 Utilisation d'octoray en tant que recepteur couple a la proteine g WO2001009166A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092624A2 (fr) * 2001-05-16 2002-11-21 Paradigm Therapeutics Limited Recepteur

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001548A2 (fr) * 1992-07-13 1994-01-20 Medical Research Council Fragments d'acide nucleiques humains isoles du cerveau, des tissus surrenaux, du placenta ou de la moelle osseuse

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001548A2 (fr) * 1992-07-13 1994-01-20 Medical Research Council Fragments d'acide nucleiques humains isoles du cerveau, des tissus surrenaux, du placenta ou de la moelle osseuse

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 17 October 1996 (1996-10-17), HILLIER ET AL., XP002934439 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092624A2 (fr) * 2001-05-16 2002-11-21 Paradigm Therapeutics Limited Recepteur
WO2002092624A3 (fr) * 2001-05-16 2003-05-08 Paradigm Therapeutics Ltd Recepteur

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