WO2000073447A1 - RinTinTin, A CHEMOKINE - Google Patents

RinTinTin, A CHEMOKINE Download PDF

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Publication number
WO2000073447A1
WO2000073447A1 PCT/US2000/014197 US0014197W WO0073447A1 WO 2000073447 A1 WO2000073447 A1 WO 2000073447A1 US 0014197 W US0014197 W US 0014197W WO 0073447 A1 WO0073447 A1 WO 0073447A1
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polypeptide
polynucleotide
sequence
seq
isolated
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PCT/US2000/014197
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French (fr)
Inventor
Edward Appelbaum
Lisa Vawter
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Smithkline Beecham Corporation
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Publication of WO2000073447A1 publication Critical patent/WO2000073447A1/en

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful m therapy, and to production of such polypeptides and polynucleotides.
  • the investigation of the critical, regulatory processes by which white cells proceed to their appropriate destination and interact with other cells is underway.
  • the current model of leukocyte movement or trafficking from the blood to injured or inflamed tissues comprises the following steps.
  • the first step is the rolling adhesion of the leukocyte along the endothehal cells of the blood vessel wall. This movement is mediated by transient interactions between selectms and their hgands.
  • a second step involves cell activation which promotes a more stable leukocyte -endothehal cell interaction mediated by the mteg ⁇ ns and their hgands. This stronger, more stable adhesion precipitates the final steps of leukocyte diapedesis and extravasation into the tissues.
  • chemokine family of polypeptide cytokmes also known as mtercrmes, possesses the cellular specificity required to explain leukocyte trafficking m different inflammatory situations
  • chemokines mediate the expression of particular adhesion molecules on endothehal cells; and second, they generate gradients of chemoattractant factors which activate specific cell types
  • the chemokines stimulate the proliferation of specific cell types and regulate the activation of cells which bear specific receptors. Both of these activities demonstrate a high degree of target cell specificity.
  • chemokines are small polypeptides, generally about 70-100 ammo acids (aa) in length, 8-11 kD in molecular weight and active over a 1-100 ng/ml concentration range Initially, they were isolated and purified from inflamed tissues and characte ⁇ zed relative to their bioactivity. More recently, chemokines have been discovered through molecular cloning techniques and characterized by structural as well as functional analysis.
  • the chemokines are related through a four cysteme motif which is based primarily on the spacing of the first two cysteme residues in the mature molecule.
  • the chemokines are assigned to one of two families, the C-X-C chemokines ( ⁇ ) and the C-C chemokines ( ⁇ ).
  • the C-X-C chemokines generally activate neutrophils and fibroblasts while the C-C chemokines act on a more diverse group of target cells which include monocytes/macrophages, basophils, eosmophils, T Lymphocytes and others.
  • the known chemokines of both families are synthesized by many diverse cell types and are reviewed in Thomson A , (1994) The Cytokme Handbook, 2d Ed. Academic Press, NY. The two groups of chemokines will be desc ⁇ bed in turn.
  • the C-C chemokines number fewer than the C-X-C chemokines, and they appear to have less N-termmal processing.
  • the macrophage inflammatory proteins alpha and beta (MlP-l ⁇ and ⁇ ) were first purified from stimulated mouse macrophage cell line and elicited an inflammatory response when injected into normal tissues.
  • At least three distinct and non-allehc genes encode human MIP- l ⁇ and seven such genes encode MlP-l ⁇ .
  • MlP-l ⁇ and MlP-l ⁇ .consist of 68-69 aa which are about 70% identical in their acidic, mature secreted forms. They are both expressed in stimulated T cells, B cells and monocytes in response to mitogens, ant ⁇ -CD3 and endotoxm , and both polypeptides bind hepa ⁇ n. While both molecules stimulate monocytes, MlP-l ⁇ chemoattracts the CD-8 subset of T lymphocytes and eosmophils, while MlP-l ⁇ chemoattracts the CD-4 subset of T lymphocytes. In mouse, these proteins are known to stimulate myelopoiesis.
  • 1-309 was cloned from a human ⁇ - ⁇ T cell line and shows 42% aa identity to T cell activation gene 3 (TCA3) cloned from mouse. There is considerable nucleotide homology between the 5' flanking regions of these two proteins, and they share an extra pair of cysteme residues not found in other chemokines. Such similarities suggest 1-309 and TCA3 are species homologs which have diverged sequence and function.
  • RANTES is another C-C chemokine which is expressed in T cells (but not B cells), in platelets, in some tumor cell lines, and in stimulated rheumatoid synovial fibroblasts. In the latter, it is regulated by mterleukms-l and -4, transforming nerve factor and mterferon- ⁇ .
  • the cDNA cloned from T cells encodes a basic 8 kD protein which lacks N-hnked glycosylation and is able to affect lymphocytes, monocytes, basophils and eosmophils. The expression of RANTES mRNA is substantially reduced following T cell stimulation.
  • Monocyte chemotactic protein (MCP-1) is a 76 aa protein which appears to be expressed in almost all cells and tissues upon stimulation by a variety of agents.
  • MCP-1 The targets of MCP-1, however, are limited to monocytes and basophils in which it induces an MCP-1 receptor, G protein-lmked calcium flux.
  • MCP-2 and MCP-3 Two other related proteins, MCP-2 and MCP-3, were purified from a human osteosarcoma cell line.
  • MCP-2 and MCP-3 have 62% and 73% a identity, respectively, with MCP- 1 and share its chemoattractant specificity for monocytes.
  • chemokine molecules are reviewed in Schall T.J., (1994) Chemotactic Cytokmes: Targets for Therapeutic Development. International Business Communications, Southborough MA pp 180-270; and m Paul W.E., (1993) Fundamental Immunology, 3rd Ed. Raven Press, NY pp 822- 826. Summary of the Invention
  • the present invention relates to RinTinTin, m particular RinTinTin polypeptides and RmTinTm polynucleotides, recombinant mate ⁇ als and methods for their production.
  • Such polypeptides and polynucleotides are of interest m relation to methods of treatment of certain diseases, including, but not limited to. allergies, aplastic anemia, asthma, atherosclerosis, autoimmune disease,
  • Behcet's disease blood transfusion reactions, chronic airway disease, carcinoma, cardiomyopathy, deep vein thrombosis, endomet ⁇ osis, g oma and other bram tumors, glomeruloneph ⁇ tis, graft vs.
  • the invention relates to methods for identifying agonists and antagonists (e g , inhibitors) using the mate ⁇ als provided by the invention, and treating conditions associated with RmTmTin imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inapprop ⁇ ate RmTinTm activity or levels.
  • the present invention relates to RinTmTm polypeptides.
  • Such polypeptides include:
  • Polypeptides of the present invention are believed to be members of the chemokine family of polypeptides. They are therefore of interest because chemokines are known to play important roles in allergic and autoimmune reactions, as well we modulating the response of the immune system to disease.
  • RmTinTm The biological properties of the RmTinTm are hereinafter referred to as "biological activity of RinTinTin” or “RmTinTm activity "
  • a polypeptide of the present invention exhibits at least one biological activity of RmTinTm.
  • Polypeptides of the present invention also includes va ⁇ ants of the aforementioned polypeptides, including all alle c 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 va ⁇ ants are those in which several, for instance 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, in any combination
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comp ⁇ sing 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 comprising 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 RinTinTin, including those with a similar activity or an improved activity, or with a decreased undesirable activity.
  • polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the lnvention.
  • 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 pu ⁇ fication, for instance multiple histidme residues, or an additional sequence for stability during recombinant production.
  • Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occu ⁇ ng sources, from genetically engineered host cells comp ⁇ sing expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods.
  • Means for preparing such polypeptides are well understood m the art.
  • the present invention relates to RinTinTin polynucleotides
  • Such polynucleotides include (a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide squence of SEQ ID NO: 1 ;
  • Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comp ⁇ smg 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 comp ⁇ smg an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
  • Preferred va ⁇ ants of polynucleotides of the present invention include splice va ⁇ ants, allehc va ⁇ ants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • Polynucleotides of the present invention also include polynucleotides encoding polypeptide va ⁇ ants that comp ⁇ se the ammo acid sequence of SEQ ID NO:2 and in which several, for instance 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 invention 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 encodmg the polypeptide of SEQ ID NO:2;
  • (b) is the RNA transc ⁇ pt of the DNA sequence encodmg 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 sequence simila ⁇ ty to genembl:al049483 [no reference].
  • the polynucleotide sequence of SEQ ID NO:l is a cDNA sequence that encodes the polypeptide of SEQ ID NO'2.
  • the polynucleotide sequence encodmg the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ID NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • polypeptide of SEQ ID NO:2 is related to other proteins of the chemokine family, having sequence similanty and/or structural simila ⁇ ty with nonred:g ⁇ 4140686 [Hromas R, Broxmeyer HE, Kim C, Ch ⁇ stopherson K 2nd, Hou YH. Biochem Biophys Res Commun;258(3):737-740].
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their similar polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one RmTinTm activity.
  • Polynucleotides of the present mvention may be obtained using standard cloning and screening techniques from a cDNA library de ⁇ ved from mRNA m cells of human placenta, (see for instance, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Sp ⁇ ng 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 libraries 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 in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem 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-histidme peptide, as provided in the pQE vector (Qiagen,
  • polynucleotide may also contain non-codmg 5 ' and 3 ' sequences, such as transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
  • 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 invention 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 similanty to SEQ ID
  • 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, including homologs from species other than human, may be obtained by a process comp ⁇ sing the steps of screening a library under st ⁇ ngent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ID NO.
  • st ⁇ ngent hyb ⁇ dization conditions include overnight incubation at 42°C in a solution comp ⁇ sing: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.
  • the present mvention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by 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.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus. This is a consequence of reverse transc ⁇ ptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template du ⁇ ng the polymerisation reaction), failing to complete a DNA copy of the mRNA template du ⁇ ng first strand cDNA synthesis.
  • the PCR reaction is then repeated using 'nested' p ⁇ mers, that is, primers designed to anneal within the amplified product (typically an adaptor specific p ⁇ mer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence).
  • primers designed to anneal within the amplified product typically an adaptor specific p ⁇ mer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence.
  • the products of this reaction can then be analysed 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 comp ⁇ sing expression systems. Accordingly, m a further aspect, the present mvention relates to expression systems comp ⁇ sing a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engineered with such expression sytems 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 invention.
  • 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 m 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, catiomc pid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • approp ⁇ ate hosts include bacte ⁇ al 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 Drosoph ⁇ a S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • bacte ⁇ al 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 Drosoph ⁇ a S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • a great va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, 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 de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage 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 that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
  • the approp ⁇ ate polynucleotide sequence may be inserted mto an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , (ibid).
  • Approp ⁇ ate secretion signals may be incorporated mto the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the pe ⁇ plasmic 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 p ⁇ or to use m the screening assay. If the polypeptide is secreted mto the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced lntracellularly, the cells must first be lysed before the polypeptide is recovered
  • Polypeptides of the present invention can be recovered and pu ⁇ fied from recombmant 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
  • high performance liquid chromatography is employed for pu ⁇ fication.
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng lntracellular synthesis, isolation and/or pu ⁇ fication.
  • Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene Detection of a mutated form of the gene characte ⁇ sed 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 in 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, u ⁇ ne, saliva, tissue biopsy or autopsy matenal.
  • 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 p ⁇ or 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. Point mutations can be identified by hybndizmg amplified DNA to labeled RmTinTm nucleotide sequences.
  • DNA sequence difference may also be detected by alterations in 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 SI protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA (1985) 85: 4397-4401).
  • An array of ohgonucleotides probes comp ⁇ smg RinTinTin 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 instance PCR, RT-PCR, RNase protection, Northern blotting and other hyb ⁇ dization 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 in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagonostic kit comprising:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transc ⁇ pt thereof;
  • kits an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO:2. It will be appreciated that m any such kit, (a), (b), (c) or (d) may comp ⁇ se a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the mvention, amongst others.
  • the polynucleotide sequences of the present invention are valuable for chromosome localization 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 mvention 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, Mendehan Inhe ⁇ tance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • DNAs Each of these DNAs contains random human genomic fragments maintained in a hamster background (human / hamster hyb ⁇ d 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 comparison is conducted at http://www.genome.wi.mit.edu/.
  • the polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present mvention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them.
  • the techniques used are well known in the art and include in situ hyd ⁇ disation techniques to clones arrayed on a g ⁇ d, such as cDNA microarray hybridisation (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 Perkin 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 mapprop ⁇ ate expression thereof in disease.
  • Such inapprop ⁇ ate expression may be of a temporal, spatial or simply quantitative nature.
  • a further aspect of the present invention relates to antibodies
  • the polypeptides of the invention or their fragments, or cells expressing 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 in the p ⁇ or art.
  • Antibodies generated against polypeptides of the present invention may be obtained by admimste ⁇ ng 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 technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hyb ⁇ doma technique (Kohler, G.
  • transgenic 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 pu ⁇ fy 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 mvention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that compnses inoculating the mammal with a polypeptide of the present mvention, 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 within the individual or not.
  • An immunological response in a mammal may also be induced by a method 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 of the invention.
  • One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
  • the formulation may further comprise a suitable carrier.
  • a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non- aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation instonic 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-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity 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 experimentation.
  • Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned. It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, in 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 invention as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, collections of chemical compounds, and natural product mixtures.
  • Such agonists or antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991)) or a small molecule.
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing 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 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 appropriate to the cells bearing the polypeptide.
  • a labeled competitor e.g agonist or antagonist
  • 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 comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a RmTinTm activity in the mixture, and comparing the RinTinTin 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 desc ⁇ bed by Schullek et al, Anal Biochem., 246, 20-29, (1997).
  • Fusion proteins such as those made from Fc portion and RmTmTm 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 m 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 in 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 binding techniques known m the art.
  • ligand binding and crosslmkmg assays include, but are not limited to, ligand binding and crosslmkmg assays in 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).
  • a source of the putative receptor 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, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • RinTinTin 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-in” animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-in 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 in 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 in the animal.
  • Transgenic animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
  • Screening kits for use in the above described methods form a further aspect of the present invention.
  • Such screening kits comprise:
  • polypeptide of the present invention an antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
  • 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 original 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 in 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 t ⁇ ple-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 metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as o gonucleotides.
  • Polypeptide refers to any polypeptide 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 refe ⁇ ed 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 that are well known m the art Such modifications are well described m 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-chains and the amino 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- ⁇ bosylation, amidation, biotmylation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylmositol, 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, lodmation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as arginylation, and ubiquitmation (see,
  • “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 polynucloetide 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 m nucleotide sequence from the reference polynucleotide.
  • nucleotide sequence of the va ⁇ ant may or may not alter the am o acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in ammo acid sequence from the reference polypeptide.
  • alterations are limited so that the sequences of the reference polypeptide and the va ⁇ ant are closely similar overall and, in many regions, identical
  • a va ⁇ ant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions in 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, lie, Leu; Asp, Glu; Asn, Gin, Ser, Thr; Lys, Arg; and Phe and Tyr.
  • a va ⁇ ant of a polynucleotide or polypeptide may be naturally occur ⁇ ng such as an allele, or it may be a va ⁇ ant 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.
  • polypeptides having one or more post-translational modifications for instance glycosylation, phosphorylation, me hylation, ADP ⁇ bosylation and the like.
  • Embodiments include methylation of the N-termmal ammo acid, phosphorylations of se ⁇ nes and threonmes and modification of C-termmal glycmes.
  • Allele refers to one of two or more alternative forms of a gene occu ⁇ ng at a given locus m the genome.
  • Polymorphism refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome withm a population.
  • SNP Single Nucleotide Polymorphism
  • SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 p ⁇ mers are required. A common primer is used in reverse complement to the polymorphism being assayed. This common p ⁇ mer can be between 50 and 1500 bps from the polymorphic base.
  • ASA Allele Specific Amplification
  • 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.
  • “Splice Va ⁇ ant” as used herein refers to cDNA molecules produced from RNA molecules initially transc ⁇ bed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
  • 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 comparing 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.
  • "similanty” means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking mto 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
  • BESTFIT is more suited to compa ⁇ ng two polynucleotide or two polypeptide sequences that are dissimilar m length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences, finding a "maximum similarity", 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 in 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 matrix (Henikoff 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 compa ⁇ son.
  • 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.
  • differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups withm the reference sequence.
  • an average of up to 5 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed.
  • a candidate polypeptide sequence having, for example, an 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- termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • an average of up to 5 in 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:l or SEQ ID NO:2, respectively
  • I is the Identity Index
  • • is the symbol for the multiplication operator, and in 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 similanty 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 m another species "Paralog” refers to a polynucleotideor polypeptide that withm the same species which is functionally similar.
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 *** discloses fusion proteins comprising various portions of constant region of lmmunoglobulm molecules together with another human protein or part thereof.
  • employing an lmmunoglobulm Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262].

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Abstract

The RinTinTin polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing RinTinTin polypeptides and polynucleotides in diagnostic assays.

Description

RinTinTin, A Chemokine
Field of the Invention
This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful m 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 as a means to identify genes and gene products as therapeutic targets is rapidly supercedmg 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 high-throughput DNA sequencing technologies and the vaπous 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 characteπze further genes and their related polypeptides/protems, as targets for drug discovery. Leukocytes including monocytes, macrophages, basophils, and eosmophils play important roles in the pathological mechanisms initiated by T and/or B lymphocytes. Macrophages, in particular, produce powerful oxidants and proteases which contribute to tissue destruction and secrete a range of cytokmes which recruit and activate other inflammatory cells.
The investigation of the critical, regulatory processes by which white cells proceed to their appropriate destination and interact with other cells is underway. The current model of leukocyte movement or trafficking from the blood to injured or inflamed tissues comprises the following steps. The first step is the rolling adhesion of the leukocyte along the endothehal cells of the blood vessel wall. This movement is mediated by transient interactions between selectms and their hgands. A second step involves cell activation which promotes a more stable leukocyte -endothehal cell interaction mediated by the mtegπns and their hgands. This stronger, more stable adhesion precipitates the final steps of leukocyte diapedesis and extravasation into the tissues. The chemokine family of polypeptide cytokmes, also known as mtercrmes, possesses the cellular specificity required to explain leukocyte trafficking m different inflammatory situations First, chemokines mediate the expression of particular adhesion molecules on endothehal cells; and second, they generate gradients of chemoattractant factors which activate specific cell types In addition, the chemokines stimulate the proliferation of specific cell types and regulate the activation of cells which bear specific receptors. Both of these activities demonstrate a high degree of target cell specificity.
The chemokines are small polypeptides, generally about 70-100 ammo acids (aa) in length, 8-11 kD in molecular weight and active over a 1-100 ng/ml concentration range Initially, they were isolated and purified from inflamed tissues and characteπzed relative to their bioactivity. More recently, chemokines have been discovered through molecular cloning techniques and characterized by structural as well as functional analysis.
The chemokines are related through a four cysteme motif which is based primarily on the spacing of the first two cysteme residues in the mature molecule. Currently, the chemokines are assigned to one of two families, the C-X-C chemokines (α) and the C-C chemokines (β). Although exceptions exist, the C-X-C chemokines generally activate neutrophils and fibroblasts while the C-C chemokines act on a more diverse group of target cells which include monocytes/macrophages, basophils, eosmophils, T Lymphocytes and others. The known chemokines of both families are synthesized by many diverse cell types and are reviewed in Thomson A , (1994) The Cytokme Handbook, 2d Ed. Academic Press, NY. The two groups of chemokines will be descπbed in turn.
At this time, the C-C chemokines number fewer than the C-X-C chemokines, and they appear to have less N-termmal processing. A brief description of human and murme C-C chemokines follows. The macrophage inflammatory proteins alpha and beta (MlP-lα and β) were first purified from stimulated mouse macrophage cell line and elicited an inflammatory response when injected into normal tissues. At least three distinct and non-allehc genes encode human MIP- lα and seven such genes encode MlP-lβ.
MlP-lα and MlP-lβ.consist of 68-69 aa which are about 70% identical in their acidic, mature secreted forms. They are both expressed in stimulated T cells, B cells and monocytes in response to mitogens, antι-CD3 and endotoxm , and both polypeptides bind hepaπn. While both molecules stimulate monocytes, MlP-lα chemoattracts the CD-8 subset of T lymphocytes and eosmophils, while MlP-lβ chemoattracts the CD-4 subset of T lymphocytes. In mouse, these proteins are known to stimulate myelopoiesis. 1-309 was cloned from a human γ-δ T cell line and shows 42% aa identity to T cell activation gene 3 (TCA3) cloned from mouse. There is considerable nucleotide homology between the 5' flanking regions of these two proteins, and they share an extra pair of cysteme residues not found in other chemokines. Such similarities suggest 1-309 and TCA3 are species homologs which have diverged sequence and function.
RANTES is another C-C chemokine which is expressed in T cells (but not B cells), in platelets, in some tumor cell lines, and in stimulated rheumatoid synovial fibroblasts. In the latter, it is regulated by mterleukms-l and -4, transforming nerve factor and mterferon-γ. The cDNA cloned from T cells encodes a basic 8 kD protein which lacks N-hnked glycosylation and is able to affect lymphocytes, monocytes, basophils and eosmophils. The expression of RANTES mRNA is substantially reduced following T cell stimulation.
Monocyte chemotactic protein (MCP-1) is a 76 aa protein which appears to be expressed in almost all cells and tissues upon stimulation by a variety of agents. The targets of MCP-1, however, are limited to monocytes and basophils in which it induces an MCP-1 receptor, G protein-lmked calcium flux. Two other related proteins, MCP-2 and MCP-3, were purified from a human osteosarcoma cell line. MCP-2 and MCP-3 have 62% and 73% aa identity, respectively, with MCP- 1 and share its chemoattractant specificity for monocytes.
Current techniques for diagnosis of abnormalities in the inflamed or diseased tissues mainly rely on observation of clinical symptoms or serological analyses of body tissues or fluids for hormones, polypeptides or various metabolites. Mammals subject to conditions or diseases associated with inflammation often manifest no clinical symptoms at early stages of disease or tumor development. Furthermore, serological analyses do not always differentiate between invasive diseases and genetic syndromes which have overlapping or very similar ranges. Thus, development of new diagnostic techniques comprising the chemokines of the present invention would provide for early and accurate diagnoses, would give a better understanding of molecular pathogenesis, and could be used m the development of effective therapies.
The chemokine molecules are reviewed in Schall T.J., (1994) Chemotactic Cytokmes: Targets for Therapeutic Development. International Business Communications, Southborough MA pp 180-270; and m Paul W.E., (1993) Fundamental Immunology, 3rd Ed. Raven Press, NY pp 822- 826. Summary of the Invention
The present invention relates to RinTinTin, m particular RinTinTin polypeptides and RmTinTm polynucleotides, recombinant mateπals and methods for their production. Such polypeptides and polynucleotides are of interest m relation to methods of treatment of certain diseases, including, but not limited to. allergies, aplastic anemia, asthma, atherosclerosis, autoimmune disease,
Behcet's disease, blood transfusion reactions, chronic airway disease, carcinoma, cardiomyopathy, deep vein thrombosis, endometπosis, g oma and other bram tumors, glomerulonephπtis, graft vs. host disease, hepatitis or other liver failures, herpes infection, malaπa, menmgioma, multiple sclerosis, osteoarthπtis, peπodontal disease, psoπasis, localized infections and septicemia, myocardial infarction, renal failure, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis and Crohn's disease, vascular diseases and Wegener's granulomatosis, hereinafter referred to as " diseases of the invention." In a further aspect, the invention relates to methods for identifying agonists and antagonists (e g , inhibitors) using the mateπals provided by the invention, and treating conditions associated with RmTmTin imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropπate RmTinTm activity or levels.
Description of the Invention
In a first aspect, the present invention relates to RinTmTm polypeptides. Such polypeptides include:
(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1 , (b) an isolated polypeptide comprising a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(c) an isolated polypeptide compπsing the polypeptide sequence of SEQ ID NO:2;
(d) an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2; (e) the polypeptide sequence of SEQ ID NO:2; and
(f) an isolated polypeptide having or compπsing 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 DO NO.2;
(g) fragments and vaπants of such polypeptides in (a) to (f).
Polypeptides of the present invention are believed to be members of the chemokine family of polypeptides. They are therefore of interest because chemokines are known to play important roles in allergic and autoimmune reactions, as well we modulating the response of the immune system to disease.
The biological properties of the RmTinTm are hereinafter referred to as "biological activity of RinTinTin" or "RmTinTm activity " Preferably, a polypeptide of the present invention exhibits at least one biological activity of RmTinTm.
Polypeptides of the present invention also includes vaπants of the aforementioned polypeptides, including all alle c forms and splice vaπants 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 vaπants are those in which several, for instance 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, in any combination
Preferred fragments of polypeptides of the present invention include an isolated polypeptide compπsing 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 comprising 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 RinTinTin, 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 corresponding full-length polypeptide by peptide synthesis; therefore, these vaπants may be employed as intermediates for producing the full-length polypeptides of the lnvention.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 puπfication, for instance multiple histidme residues, or an additional sequence for stability during recombinant production.
Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occuπng sources, from genetically engineered host cells compπsing expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods.. Means for preparing such polypeptides are well understood m the art.
In a further aspect, the present invention relates to RinTinTin polynucleotides Such polynucleotides include (a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide squence of SEQ ID NO: 1 ;
(b) an isolated polynucleotide compπsing the polynucleotide of SEQ ID NO: 1 ,
(c) an isolated polynucleotide having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide of SEQ ID NO: 1 ;
(d) the isolated polynucleotide of SEQ ID NO: 1 ;
(e) an isolated polynucleotide compπsing 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;
(f) an isolated polynucleotide compπsing a polynucleotide sequence encodmg the polypeptide of SEQ ID NO:2;
(g) an isolated polynucleotide having 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;
(h) an isolated polynucleotide encodmg the polypeptide of SEQ ID NO:2;
(l) an isolated polynucleotide having or comprising a polynucleotide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polynucleotide sequence of SEQ ID NO:l;
(j) an isolated polynucleotide having or comprising a polynucleotide sequence encoding 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; and polynucleotides that are fragments and variants 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 invention include an isolated polynucleotide compπsmg 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 compπsmg an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
Preferred vaπants of polynucleotides of the present invention include splice vaπants, allehc vaπants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
Polynucleotides of the present invention also include polynucleotides encoding polypeptide vaπants that compπse the ammo acid sequence of SEQ ID NO:2 and in which several, for instance 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
In a further aspect, the present invention provides polynucleotides that are RNA transcπpts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that'
(a) comprises an RNA transcπpt of the DNA sequence encodmg the polypeptide of SEQ ID NO:2;
(b) is the RNA transcπpt of the DNA sequence encodmg the polypeptide of SEQ ID NO:2;
(c) comprises an RNA transcπpt of the DNA sequence of SEQ ID NO: 1 ; or (d) is the RNA transcπpt 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 sequence similaπty to genembl:al049483 [no reference]. The polynucleotide sequence of SEQ ID NO:l is a cDNA sequence that encodes the polypeptide of SEQ ID NO'2. The polynucleotide sequence encodmg the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ID NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide of SEQ ID NO:2 is related to other proteins of the chemokine family, having sequence similanty and/or structural similaπty with nonred:gι4140686 [Hromas R, Broxmeyer HE, Kim C, Chπstopherson K 2nd, Hou YH. Biochem Biophys Res Commun;258(3):737-740].
Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their similar polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one RmTinTm activity. Polynucleotides of the present mvention may be obtained using standard cloning and screening techniques from a cDNA library deπved from mRNA m cells of human placenta, (see for instance, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries 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 in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions For example, a marker sequence that 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 Natl Acad Sci 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 mRNA. Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of
SEQ ID NO.1 , may be used as hybπdization probes for cDNA and genomic DNA or as pπmers for a nucleic acid amplification reaction (for instance, PCR) Such probes and pπmers may be used 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 paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similanty to SEQ ID
NO: 1 , typically at least 95% identity. Preferred probes and pπmers will generally compπse 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 pπmers will have between 20 and 25 nucleotides. A polynucleotide encoding a polypeptide of the present invention, including homologs from species other than human, may be obtained by a process compπsing the steps of screening a library under stπngent hybπdization 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. Such hybπdization techniques are well known to the skilled artisan. Preferred stπngent hybπdization conditions include overnight incubation at 42°C in a solution compπsing: 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. lx SSC at about 65°C. Thus the present mvention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides.
The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus. 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 duπng the polymerisation reaction), failing to complete a DNA copy of the mRNA template duπng first strand cDNA synthesis.
There are several methods available and well known to those skilled m 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., Proc Nat Acad Sci USA 85, 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon (trade mark) technology (Clontech Laboratoπes Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon (trade mark) technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated 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' pπmers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pπmer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence). The products of this reaction can then be analysed 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, m a further aspect, the present mvention relates to expression systems compπsing a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engineered with such expression sytems 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. Polynucleotides may be introduced into host cells by methods descπbed m 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, catiomc pid-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 Drosophύa 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-deπved 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 that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropπate polynucleotide sequence may be inserted mto an expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , (ibid). Appropπate secretion signals may be incorporated mto 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 mto the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced lntracellularly, 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 interaction 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 lntracellular synthesis, isolation and/or puπfication.
Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene Detection of a mutated form of the gene characteπsed 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 in the gene may be detected at the DNA level by a vaπety of techniques well known in the art
Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, uπne, saliva, tissue biopsy or autopsy matenal. 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 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 in compaπson to the normal genotype. Point mutations can be identified by hybndizmg amplified DNA to labeled RmTinTm nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denatuπng 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 SI protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA (1985) 85: 4397-4401). An array of ohgonucleotides probes compπsmg RinTinTin 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 gπds. 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 vaπabihty, 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 instance PCR, RT-PCR, RNase protection, Northern blotting and other hybπdization 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 in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Thus in another aspect, the present invention relates to a diagonostic kit comprising:
(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transcπpt thereof;
(b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present mvention, preferably the polypeptide of SEQ ID NO.2 or a fragment thereof; or
(d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO:2. It will be appreciated that m 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 diseases of the mvention, amongst others.
The polynucleotide sequences of the present invention are valuable for chromosome localization studies. 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 mvention 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, Mendehan 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 (co- lnheπtance of physically adjacent genes). Precise human chromosomal localisations for a genomic sequence (gene fragment etc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) A method for constructing radiation hybπd maps of whole genomes, Nature Genetics 7, 22-28). A number of RH panels are available from Research Genetics (Huntsville, AL, USA) e.g. the GeneBπdge4 RH panel (Hum Mol Genet 1996 Mar;5(3):339-46 A radiation hybπd map of the human genome. Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet D, Prud'Homme JF, Dib C, Auffray C, Moπssette J, Weissenbach J, Goodfellow PN). To determine the chromosomal location of a gene using this panel, 93 PCRs are performed using pπmers 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 hybπd 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 comparison is conducted at http://www.genome.wi.mit.edu/.
The polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present mvention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include in situ hydπdisation techniques to clones arrayed on a gπd, such as cDNA microarray hybridisation (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 Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of mappropπate expression thereof in disease. Such inappropπate expression may be of a temporal, spatial or simply quantitative nature.
A further aspect of the present invention relates to antibodies The polypeptides of the invention or their fragments, or cells expressing 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 in the pπor art.
Antibodies generated against polypeptides of the present invention may be obtained by admimsteπng the polypeptides or epitope-beaπng fragments, 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 Milstein, 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, 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 smgle 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 diseases of the invention, amongst others.
Polypeptides and polynucleotides of the present mvention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that compnses inoculating the mammal with a polypeptide of the present mvention, 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 within the individual or not. An immunological response in a mammal may also be induced by a method 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 of the invention. One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid. For use a vaccine, 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 intradermal injection). Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation instonic 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-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity 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 experimentation.
Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned. It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, in 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 invention as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, collections of chemical compounds, and natural product mixtures. Such agonists or antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991)) or a small molecule. The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing 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 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 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 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 a RmTinTm activity in the mixture, and comparing the RinTinTin 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. Such 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 descπbed by Schullek et al, Anal Biochem., 246, 20-29, (1997).
Fusion proteins, such as those made from Fc portion and RmTmTm polypeptide, as hereinbefore descπbed, 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 mRNA 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 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 binding techniques known m 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, * ->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 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.
Examples of antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
Screening methods may also involve the use of transgenic technology and RinTinTin gene. The art of constructing transgenic animals is well established. For example, the RinTinTin 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-in" animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-in transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target. Other useful 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 in 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 in the animal. Transgenic animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
Screening kits for use in the above described methods form a further aspect of the present invention. Such screening kits comprise:
(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) an antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
Glossary 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 its natural state, i e , if it occurs in 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 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. Moreover, 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 in said organism, which organism may be living or non-living.
"Polynucleotide" generally refers to any polyπbonucleotide (RNA) or polydeoxπbonucleotide (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. 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 include, for example, tπtylated bases and unusual bases such as inosme. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as o gonucleotides.
"Polypeptide" refers to any polypeptide 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 refeπed 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 that are well known m the art Such modifications are well described m 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-chains and the amino 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, biotmylation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylmositol, 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, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as arginylation, 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, 1-12, in Post-translational 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, 182, 626-646, 1990, and Rattan et al., "Protein Synthesis: Post-translational Modifications and Agmg", Ann NY Acad Sci, 663, 48-62, 1992).
"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 polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO: 1..
"Vaπant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical vaπant of a polynucleotide differs m nucleotide sequence from the reference polynucleotide. Changes m the nucleotide sequence of the vaπant may or may not alter the am o acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in ammo acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the vaπant are closely similar overall and, in many regions, identical A vaπant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions in 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, lie, Leu; Asp, Glu; Asn, Gin, Ser, Thr; Lys, Arg; and Phe and Tyr. A vaπant of a polynucleotide or polypeptide may be naturally occurπng such as an allele, or it may be a vaπant 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. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, me hylation, ADP πbosylation and the like. Embodiments include methylation of the N-termmal ammo acid, phosphorylations of seπnes and threonmes and modification of C-termmal glycmes.
"Allele" refers to one of two or more alternative forms of a gene occuπng at a given locus m the genome.
"Polymorphism" refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome withm a population.
"Single Nucleotide Polymorphism" (SNP) refers to the occurence of nucleotide variability at a single nucleotide position in the genome, withm a population. An SNP may occur withm a gene or within lntergemc regions of the genome. SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 pπmers are required. A common primer is used in reverse complement to the polymorphism being assayed. This common pπmer can be between 50 and 1500 bps from the polymorphic base. The other two (or more) pπmers 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. "Splice Vaπant" as used herein refers to cDNA molecules produced from RNA molecules initially transcπbed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcπpt 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 comparing 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 In general, 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. In general, "similanty" means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking mto 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.
Methods for comparing the identity and similarity of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similaπty between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similarity between two sequences. BESTFIT is more suited to compaπng two polynucleotide or two polypeptide sequences that are dissimilar m length, the program assuming that the shorter sequence represents a portion of the longer. In companson, GAP aligns two sequences, finding a "maximum similarity", according to the algoπthm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to compaπng sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters "Gap Weight" and "Length Weight" used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively Preferably, % identities and similaπties are determined when the two sequences being compared are optimally aligned. Other programs for determining identity and or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, J Mol Biol, 215, 403-410, 1990, Altschul S F et al, Nucleic Acids Res., 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA and accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85,
2444-2448,1988, available as part of the Wisconsin Sequence Analysis Package).
Preferably, the BLOSUM62 ammo acid substitution matrix (Henikoff 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 compaπson.
Preferably, 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 descπbed. "Identity Index" is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence. Thus, for instance, 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. These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups withm the reference sequence. In other words, to obtain a polynucleotide sequence having an Identity Index of 0.95 compared to a reference polynucleotide sequence, an average of up to 5 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descπbed. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99. Similarly, for a polypeptide, a candidate polypeptide sequence having, for example, an 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- termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polypeptide sequence having an Identity Index of 0.95 compared to a reference polypeptide sequence, an average of up to 5 in every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descπbed. The same applies mutatis mutandis for other values of the Identity Index, for instance 0 96, 0.97, 0.98 and 0.99.
The relationship between the number of nucleotide or ammo acid differences and the Identity Index may be expressed m the following equation.
na < xa - (xa • I),
in which: na is the number of nucleotide or ammo acid differences, xa is the total number of nucleotides or ammo acids in SEQ ID NO:l or SEQ ID NO:2, respectively,
I is the Identity Index ,
• is the symbol for the multiplication operator, and in which any non-mteger product of xa and I is rounded down to the nearest integer pπor to subtracting it from xa.
"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 similanty between the two sequences as hereinbefore defined. Falling withm this geneπc term are the terms "ortholog", and "paralog". "Ortholog" refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide m another species "Paralog" refers to a polynucleotideor polypeptide that withm 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 *** discloses fusion proteins comprising various portions of constant region of lmmunoglobulm molecules together with another human protein or part thereof. In many cases, employing an lmmunoglobulm Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in, for example, improved pharmacokinetic 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 and references, including but not limited to patents and patent applications, cited this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner descπbed above for publications and references.

Claims

What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1 ; (b) an isolated polypeptide comprising a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2;
(c) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO:2;
(d) an isolated polypeptide having at least 95% identity to the polypeptide sequence of SEQ ID NO.2;
(e) the polypeptide sequence of SEQ ID NO.2; and (f) fragments and variants of such polypeptides in (a) to (e)
2. An isolated polynucleotide selected from the group consisting of:
(a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95% identity to the polynucleotide squence of SEQ ID NO: 1 ; (b) an isolated polynucleotide compπsing the polynucleotide of SEQ ID NO: 1 ;
(c) an isolated polynucleotide having at least 95% identity to the polynucleotide of SEQ ID NO: 1 ;
(d) the isolated polynucleotide of SEQ ID NO: 1 ;
(e) an isolated polynucleotide compπsing a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2; (f) an isolated polynucleotide compns g a polynucleotide sequence encodmg the polypeptide of SEQ
ID NO:2;
(g) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2;
(h) an isolated polynucleotide encodmg the polypeptide of SEQ ID NO:2; (l) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides obtained by screenmg a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof having at least 15 nucleotides; and
0) a polynucleotide which is the RNA equivalent of a polynucleotide of (a) to (I); or a polynucleotide sequence complementary to said isolated polynucleotide and polynucleotides that are vaπants and fragments of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof
3. An antibody lmmunospecific for the polypeptide of claim 1.
4. An antibody as claimed in claim 3 which is a polyclonal antibody.
5. An expression vector compπsing a polynucleotide capable of producing a polypeptide of claim 1 when said expression vector is present in a compatible host cell.
6. A process for producing a recombinant host cell which comprises the step of introducing an expression vector compπsmg a polynucleotide capable of producing a polypeptide of claim 1 mto a cell such that the host cell, under appropriate culture conditions, produces the polypeptide of claim 1.
7. A recombinant host cell produced by the process of claim 6.
8. A membrane of the recombinant host cell of claim 7 expressing the polypeptide of claim 1.
9. A process for producing a polypeptide which comprises cultuπng the host cell of claim 8 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.
PCT/US2000/014197 1999-05-28 2000-05-24 RinTinTin, A CHEMOKINE WO2000073447A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023750A2 (en) * 1996-11-27 1998-06-04 Schering Corporation Mammalian chemokines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023750A2 (en) * 1996-11-27 1998-06-04 Schering Corporation Mammalian chemokines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE ON DIALOG, US NATIONAL LIBRARY OF MEDICINE, (BETHESDA, MD, USA); HEDRICK ET AL.: "DVic-1 and DGWCC chemokines useful for developing products for treating abnormal physiology or development e.g. cancerous or degenerative conditions", XP002931870 *
H. ROMAS ET AL.: "Isolation of ALP, a novel divergent murine CC chemokine with a unique carboxy terminal extension", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,, vol. 258, 1999, pages 737 - 740, XP002931869 *

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