WO2003104278A1 - Proteine de type tnf - Google Patents

Proteine de type tnf Download PDF

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Publication number
WO2003104278A1
WO2003104278A1 PCT/GB2003/002510 GB0302510W WO03104278A1 WO 2003104278 A1 WO2003104278 A1 WO 2003104278A1 GB 0302510 W GB0302510 W GB 0302510W WO 03104278 A1 WO03104278 A1 WO 03104278A1
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WIPO (PCT)
Prior art keywords
polypeptide
nucleic acid
disease
ofthe
tnf
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PCT/GB2003/002510
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English (en)
Inventor
Christine Power
Richard Joseph Fagan
Christopher Benjamin Phelps
Richard James Mitter
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Ares Trading S.A.
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Priority to AU2003240085A priority Critical patent/AU2003240085A1/en
Publication of WO2003104278A1 publication Critical patent/WO2003104278A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to a protein, termed BAB71417.1 herein identified as being a novel member ofthe TNF (tumor necrosis factor)-like family of cytokines and to the use of this protein and nucleic acid sequence from the encoding gene in the diagnosis, prevention and treatment of disease.
  • TNF tumor necrosis factor
  • a protein whose protein sequence is recorded in the public NCBI database as NCBI nucleotide AK057304.1 and NCBI protein BAB71417.1 is implicated as a novel member ofthe TNF-like family of cytokines.
  • Cytokines are a family of growth factors secreted primarily from leukocytes, and are messenger proteins that act as potent regulators capable of effecting cellular processes at sub-nanomolar concentrations. Interleukins, neurotrophins, growth factors, interferons and chemokines all define cytokine families that work in conjunction with cellular receptors to regulate cell proliferation and differentiation. Their size allows cytokines to be quickly transported around the body and degraded when required. Their role in controlling a wide range of cellular functions, especially the immune response and cell growth has been revealed by extensive research over the last twenty years (Boppana, S.B (1996) Indian. J. Pediatr. 63(4):447-52).
  • Cytokines as for other growth factors, are differentiated from classical hormones by the fact that they are produced by a number of different cell types rather than just one specific tissue or gland, and also effect a broad range of cells via interaction with specific high affinity receptors located on target cells. All cytokine communication systems show both pleiotropy (one messenger producing multiple effects) and redundancy (each effect is produced by more than one messenger (Tringali, G. et al, (2000) Therapie. 55(l):171-5; Tessarollo, L. (1998) Cytokine Growth Factor Rev. 9(2):125-137). An individual cytokine's effects on a cell can also be dependent on its concentration, the concentration of other cytokines, the temporal sequence of cytokines, and the internal state of the cell (cell cycle, presence of neighbouring cells, cancerous).
  • cytokines are typically small (under 200 amino acids) proteins they are often formed from larger precursors which are post-translationally spliced. This, in addition to mRNA alternative splicing pathways, give a wide spectrum of variants of each cytokine, each of which may differ substantially in biological effect. Membrane and extracellular matrix associated forms of many cytokines have also been isolated (Okada-Ban, M. et al, (2000) Int. J. Biochem. Cell Biol. 32(3):263-267; Atamas, S.P. (1997) Life Sci. 61(12):1105-1112).
  • Cytokines can be grouped into families, though most are unrelated. Categorisation is usually based on secondary structure composition, as sequence similarity is often very low. The families are named after the archetypal member e.g. IFN-like, IL2-like, ILl-like, IL-6 like and TNF-like (Zlotnik, A. et al, (2000) Immunity. 12(2):121-127).
  • cytokines are involved in many important reactions in multi-cellular organisms such as immune response regulation ( ⁇ ishihira, J. (1998) Int. J. Mol. Med. 2(1): 17-28), inflammation (Kim, P.K. et al, (2000) Surg. Clin. North. Am. 80(3):885-894), wound healing (Clark, R.A. (1991) J. Cell Biochem. 46(1): 1-2), embryogenesis and development, and apoptosis (Flad, H.D. et al, (1999) Pathobiology. 67(5-6):291-293).
  • Pathogenic organisms such as FUN and Kaposi's sarcoma- associated virus encode anti-cytokine factors as well as cytokine analogues, which allow them to interact with cytokine receptors and control the body's immune response (Sozzani, S. et al, (2000) Pharm. Acta. Helv. 74(2-3):305-312; Aoki, Y. et al, (2000) J. Hematother. Stem Cell Res. 9(2): 137-145). Nirally encoded cytokines, virokines, have been shown to be required for pathogenicity of viruses due to their ability to mimic and subvert the host immune system.
  • Cytokines may be useful for the treatment, prevention and/or diagnosis of medical conditions and diseases including cell proliferative disorders, autoimmune/iiiflammatory disorders, genetic disorders, developmental disorders, nervous system disorders, metabolic disorders, infections and other pathological conditions; particularly immune disorders, such as autoimmune disease, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, and multiple sclerosis, inflammatory disorders, such as allergy, rhinitis, conjunctivitis, glomerulonephritis, uveitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, pancreatitis, digestive system inflammation, sepsis, endotoxic shock, septic shock, cachexia, myalgia, ankylosing spondylitis, myasthenia gravis, post-viral fatigue syndrome, pulmonary disease, respiratory distress syndrome, asthma, chronic-obstructive pulmonary disease, airway inflammation, wound healing, endometri
  • cytokines have focused on their role as regulators of the immune system (Rodriguez, F.H. et al, (2000) Curr. Pharm. Des. 6(6):665-680) for instance in promoting a response against thyroid cancer (Schmutzler, C. et al, (2000) 143(l):15-24). Their control of cell growth and differentiation has also made cytokines anti-cancer targets (Lazar- Molnar, E. et al, (2000) Cytokine. 12(6):547-554; Gado, K. (2000) 24(4): 195-209). Novel mutations in cytokines and cytokine receptors have been shown to confer disease resistance in some cases (van Deventer, SJ.
  • TNF alpha and beta are examples of cytokines, which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease.
  • the TNF molecules belong to the "TNF-ligand” superfamily, and act together with their receptors or counter-ligands, the "TNF-receptor” superfamily. So far, a number of members of the TNF ligand superfamily have been identified along and several members ofthe TNF-receptor superfamily have been characterized.
  • TNF-alpha lymphotoxin-alpha
  • LT-alpha lymphotoxin-alpha
  • LT-beta lymphotoxin-alpha
  • FasL CD40L
  • CD27L CD30L
  • 4-1BBL 4-1BBL
  • OX40L nerve growth factor
  • NGF nerve growth factor
  • the superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals 22:291-295 (1994)). Many members ofthe TNF-ligand superfamily are expressed by activated T-cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (Meager, A., supra).
  • cytokines such as TNF-like molecules
  • TNF-like molecules provide a means to alter disease phenotype and as such, identification of novel cytokines is highly relevant as they may play a role in or be useful in the development of treatments for the diseases identified above, as well as other disease states.
  • the invention is based on the discovery that the BAB71417.1 protein functions as a TNF- like protein.
  • the BAB71417.1 protein it has been found that a region including residues 29-127 of this protein sequence adopts an equivalent structure to residues 34-144 of Human TNF_beta (PDB code 1TNRA).
  • TNF_beta is known to function as a TNF-like protein. This relationship is not just to TNF_beta, but rather to the TNF-like protein family as a whole.
  • the discovery of sharing an equivalent region allows the functional annotation of this region of BAB71417.1, a d therefore proteins that include this region, as possessing TNF-like protein activity.
  • the invention provides a polypeptide, which polypeptide: (i) comprises the amino acid sequence as recited in SEQ ID NO:2;
  • (ii) is a fragment thereof having a TNF-like protein activity or having an antigenic determinant in common with the polypeptides of (i); or
  • a polypeptide according to the first aspect of the invention consists of the amino acid sequence as recited in SEQ ID NO:2.
  • polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "the BAB71417.1 polypeptide”.
  • a preferred polypeptide fragment according to part ii) above includes the region ofthe BAB71417.1 polypeptide that is predicted as that responsible for TNF-like protein activity (hereafter, the "BAB71417.1 TNF-like protein region"), or is a variant thereof.
  • the BAB71417.1 TNF-like protein region is considered to extend between residue 29 and residue 127 of the BAB71417.1 polypeptide sequence.
  • This aspect of the invention also includes fusion proteins that incorporate polypeptide fragments and variants of these polypeptide fragments as defined above, provided that said fusion proteins possess activity as a TNF-like protein.
  • the invention provides a purified nucleic acid molecule that encodes a polypeptide of the first aspect of the invention.
  • the purified nucleic acid molecule has the nucleic acid sequence as recited in SEQ ID NO:l (encoding the BAB71417.1 polypeptide), or is a redundant equivalent or fragment of this sequence.
  • a preferred nucleic acid fragment is one that encodes a polypeptide fragment according to part ii) above, preferably a polypeptide fragment that includes the BAB71417.1 TNF-like protein region, or that encodes a variant of these fragments as this term is defined above.
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of tlie invention.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule ofthe second or third aspect ofthe invention.
  • the invention provides a host cell transformed with a vector ofthe fourth aspect ofthe invention.
  • the invention provides a ligand which binds specifically to, and which preferably inhibits the TNF-like protein activity of, a polypeptide ofthe first aspect ofthe invention.
  • Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substarates, enzymes, receptors, small organic moleucles such as small natural or synthetic organic moleucle of up to 2000 Da, preferably 800 Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics ofthe aforementioned.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide ofthe first aspect ofthe invention.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification ofthe function ofthe region defined herein as the TNF-like protein region of the BAB71417.1 polypeptide allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of diseases in which TNF-like proteins are implicated.
  • the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis of disease.
  • TNF-like proteins examples include cell proliferative disorders, autoimmune/inflammatory disorders, genetic disorders, developmental disorders, nervous system disorders, metabolic disorders, infections and other pathological conditions; particularly immune disorders, such as autoimmune disease, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, and multiple sclerosis, inflammatory disorders, such as allergy, rhinitis, conjunctivitis, glomerulonephritis, uveitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, pancreatitis, digestive system inflammation, sepsis, endotoxic shock, septic shock, cachexia, myalgia, ankylosing spondylitis, myasthenia gravis, post-viral fatigue syndrome, pulmonary disease, respiratory distress syndrome, asthma, chronic-obstructive pulmonary disease, airway inflammation, wound healing, endometriosis, dermatological disease, Behcet's
  • These molecules may also be used in the manufacture of a medicament for the treatment of such diseases.
  • These moieties ofthe first, second, third, fourth, fifth, sixth or seventh aspect of the invention may also be used in the manufacture of a medicament for the treatment of such diseases.
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient.
  • the invention also provides kits that are useful in these methods for diagnosing disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, ofthe sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • a ligand such as an antibody
  • PCR polymerase chain reaction
  • the disease diagnosed by a method of the ninthaspect of the inventio is a disease in which members ofthe TNF-like protein family of cytokines are implicated, as described above.
  • the invention provides for the use of a polypeptide ofthe first aspect of the invention, or a fragment thereof, as a TNF-like protein.
  • the invention also provides for the use of a nucleic acid molecule according to the second or third aspects of the invention to express a protein that possesses TNF-like activity.
  • the invention also provides a method for effecting TNF-like protein activity, said method utilising a polypeptide ofthe first aspect ofthe invention.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect ofthe invention, or a vector ofthe fourth aspect ofthe invention, or a host cell according to the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically-acceptable carrier.
  • the present invention provides a polypeptide ofthe first aspect ofthe invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector ofthe fourth aspect ofthe invention, or a host cell according to the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease in which TNF-like proteins are implicated, examples of which are listed above.
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide ofthe first aspect ofthe invention, or a nucleic acid molecule ofthe second or third aspect ofthe invention, or a vector ofthe fourth aspect ofthe invention, or a host cell according to the fifth aspect ofthe invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, vector, host cell, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be using in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • the disease is a disease in which members of TNF-like proteins are implicated, as described above.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide ofthe present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification ofthe mature polypeptide sequence.
  • the polypeptide ofthe first aspect ofthe invention may form part of a fusion protein.
  • additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
  • modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage ofthe amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides ofthe present invention.
  • modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the BAB71417.1 polypeptide.
  • Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity” indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity” indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford
  • Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) ofthe BAB71417.1 polypeptide.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more ofthe amino acid residues includes a substituent group.
  • polypeptides of the first aspect ofthe invention have a degree of sequence identity with the BAB71417.1 polypeptide, or with active fragments thereof, of greater than 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively with the BAB71417.1 polypeptide, or with active fragments thereof.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome ThreaderTM technology that forms one aspect of the search tools used to generate the Biopendium search database may be used to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the BAB71417.1 polypeptide, are predicted to have TNF-like activity, by virtue of sharing significant structural homology with the BAB71417.1 polypeptide sequence.
  • the Inpharmatica Genome ThreaderTM predicts two proteins, or protein regions, to share structural homology with a certainty of at least 10% more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and above.
  • the certainty value of the Inpharmatica Genome ThreaderTM is calculated as follows. A set of comparisons was initially performed using the Inpharmatica Genome ThreaderTM exclusively using sequences of known structure. Some of the comparisons were between proteins that were known to be related (on the basis of structure). A neural network was then trained on the basis that it needed to best distinguish between the known relationships and known not-relationships taken from the CATH structure classification (www.biochem.ucl.ac.uk/bsm/cath).
  • TNF-like protein region BAB71417.1 TNF-like protein region.
  • Such structural homologues are predicted to have TNF-like protein activity by virtue of sharing significant structural homology with this polypeptide sequence.
  • polypeptides of the first aspect of the invention also include fragments of the BAB71417.1 polypeptide, functional equivalents of the fragments of the BAB71417.1 polypeptide, and fragments of the functional equivalents of the BAB71417.1 polypeptides, provided that those functional equivalents and fragments retain TNF-like protein activity or have an antigenic determinant in common with the BAB71417.1 polypeptide.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the BAB71417.1 polypeptides or one of its functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
  • Preferred polypeptide fragments according to this aspect of the invention are fragments that include a region defined herein as the TNF-like protein region of the BAB71417.1 polypeptides, respectively. These regions are the regions that have been annotated as BAB71417.1 TNF -like protein region.
  • this region is considered to extend between residue 29 and residue 127.
  • Variants of this fragment are included as embodiments of this aspect of the invention, provided that these variants possess activity as a TNF-like protein.
  • variable is meant to include extended or truncated versions of this polypeptide fragment.
  • the BAB71417.1 TNF -like protein region of the BAB71417.1 polypeptide will fold correctly and show TNF-like protein activity if additional residues C terminal and/or N terminal of these boundaries in the BAB71417.1 polypeptide sequence are included in the polypeptide fragment.
  • additional 5, 10, 20, 30, 40 or even 50 or more amino acid residues from the BAB71417.1 polypeptide sequence, or from a homologous sequence may be included at either or both the C terminal and/or N terminal ofthe boundaries ofthe TNF-like protein regions of the BAB71417.1 polypeptide, without prejudicing the ability of the polypeptide fragment to fold correctly and exhibit TNF-like protein activity.
  • one or more amino acid residues may be deleted at either or both the C terminus or the N terminus of the TNF-like protein region of the BAB71417.1 polypeptide.
  • variant includes homologues ofthe polypeptide fragments described above, that possess significant sequence homology with the TNF-like protein region of the BAB71417.1 polypeptide, provided that said variants retain activity as a TNF-like protein.
  • variant homologues of polypeptide fragments of this aspect of the invention have a degree of sequence identity with the TNF-like protein regions of the BAB71417.1 polypeptides, of greater than 85%.
  • variant polypeptides have degrees of identity of greater than 90%, 95%, 98% or 99%, respectively with the TNF-like protein regions of the BAB71417.1 polypeptides, provided that said variants retain activity as a TNF-like protein.
  • variant polypeptides also include homologues ofthe truncated forms ofthe polypeptide fragments discussed above, provided that said variants retain activity as a TNF-like protein.
  • polypeptide fragments of the first aspect of the invention may be polypeptide fragments that exhibit significant structural homology with the structure of the polypeptide fragment defined by the TNF-like protein regions of the BAB71417.1 polypeptide sequence, for example, as identified by the Inpharmatica Genome ThreaderTM. Accordingly, polypeptide fragments that are structural homologues of the polypeptide fragments defined by the BAB71417.1 A TNF-like protein region of the BAB71417.1 polypeptide sequence should adopt the same fold as that adopted by this polypeptide fragment, as this fold is defined above.
  • fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the fragment ofthe invention When comprised within a larger polypeptide, the fragment ofthe invention most preferably forms a single continuous region.
  • certain preferred embodiments relate to a fragment having a pre - and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment.
  • several fragments may be comprised within a single larger polypeptide.
  • polypeptides ofthe present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides.
  • ligands such as polyclonal or monoclonal antibodies
  • Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
  • the antibodies may also be employed as diagnostic or therapeutic aids, amongst otlier applications, as will be apparent to the skilled reader.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect ofthe invention.
  • substantially greater affinity we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known cell- surface receptors.
  • the affinity is at least 1.5-fole, 2-fold, 5-fold, 10-fold, 100-fold, 10 3 -fold, 10 4 - fold, 10 5 -fold, 10 6 -fold or greater for a polypeptide of the invention than for known cell- surface receptor polypeptides.
  • a selected mammal such as a mouse, rabbit, goat or horse, may be immunised with a polypeptide of the first aspect of the invention.
  • the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically. If desired, the polypeptide can be conjugated to a carrier protein.
  • polypeptides may be chemically coupled.
  • the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography.
  • Monoclonal antibodies to the polypeptides ofthe first aspect ofthe invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G.
  • Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
  • Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
  • the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239: 1534 (1988); Kabat et al, J. Immunol., 147: 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci.
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains ofthe heavy and/or light chains of a non-human donor antibody have been substituted in place ofthe equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability ofthe donor antibody.
  • the antibody may be a "bispecific" antibody, that is an antibody having two different antigen-binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCaf erty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode the polypeptide sequences recited in SEQ ID NO:2, and functionally equivalent polypeptides, including active fragments of the BAB71417.1 polypeptide, such as a fragment including the TNF-like protein region ofthe BAB71417.1 polypeptide sequence, or a homologue thereof. Nucleic acid molecules encompassing these stretches of sequence form a preferred embodiment of this aspect ofthe invention.
  • nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.
  • the nucleic acid molecules may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • nucleic acid molecule also includes analogues of DNA and RNA, such as those containing modified backbones ,and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:2, or an active fragment thereof may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:l. These molecules also may have a different sequence wliich, as a result of the degeneracy of the genetic code, encodes the polypeptide SEQ ID NO:2, or an active fragment of the BAB71417.1 polypeptide, such as a fragment including the BAB71417.1 TNF-like protein region, or a homologue thereof.
  • the BAB71417.1 TNF- like protein region is considered to extend between residue 29 and residue 127 of the BAB71417.1 polypeptide sequence.
  • the BAB71417.1 TNF-like protein region is thus encoded by a nucleic acid molecule including nucleotide 85 to nucleotide 381.
  • nucleic acid molecules that encode the polypeptide of SEQ ID NO:2 may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3* sequences, such as the transcribed, non- translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
  • the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
  • the nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect ofthe invention.
  • a preferred fragment of the BAB71417.1 polypeptide is a fragment including the BAB71417.1 TNF-like protein region, or a variant or homologue thereof, as these terms are defined above.
  • the BAB71417.1 TNF-like protein region is encoded by a nucleic acid molecule including nucleotide 85 to nucleotide 381 of SEQ ID NO : 1.
  • nucleic acid molecules according to the invention may be naturally-occurring variants such as a naturally-occurring allelic variant, or the molecules may be a variant that is not known to occur naturally.
  • Such non-naturally occurring variants ofthe nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions.
  • the substitutions, deletions or insertions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
  • the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
  • Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
  • antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide ofthe invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. [supra]).
  • the inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al [supra]).
  • a substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCI, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • Preferred embodiments of this aspect ofthe invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the BAB71417.1 polypeptide (SEQ ID NO:2), and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a preferred active fragment is a fragment that includes the TNF-like protein region of the BAB71417.1 polypeptide sequences, respectively.
  • preferred nucleic acid molecules include those that are at least 70% identical over their entire length to a nucleic acid molecule encoding the TNF-like protein region of the B AB71417.1 polypeptide sequence.
  • Percentage identity is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/).
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to the nucleic acid molecule having the sequence given in SEQ ID NO:l, to a region including nucleotides 85-381 of this sequence or a nucleic acid molecule that is complementary to any of these regions of nucleic acid.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the BAB71417.1 polypeptide.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the BAB71417.1 polypeptide and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the BAB71417.1 polypeptide, particularly with an equivalent function to the TNF-like protein region of the BAB71417.1 polypeptide is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:l), particularly a region from nucleotides 85-381, or from nucleotides 85-381 of SEQ ID NO : 1 , are particularly useful probes.
  • Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al., Proc. Natl. Acad. Sci. USA (1988) 85: 8998-9002).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1: 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055- 3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation.
  • a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the 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, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation.
  • Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.
  • RNA interference (Elbashir, SM et al, Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.
  • the vectors ofthe present invention comprise nucleic acid molecules ofthe invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfested or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides ofthe invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those described in Sambrook et al, (supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • Human artificial chromosomes may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
  • Cell-free translation systems can also be employed to produce the polypeptides ofthe invention.
  • nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs ofthe system.
  • the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion ofthe translated polypeptide into the lumen ofthe endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
  • a control sequence such as a signal peptide or leader sequence
  • These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Leader sequences can be removed by the bacterial host in post-translational processing.
  • regulatory sequences that allow for regulation ofthe expression ofthe polypeptide relative to the growth ofthe host cell.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Sfratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • stable expression is preferred.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene.
  • Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
  • Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.
  • yeast cells for example, S. cerevisiae
  • Aspergillus cells examples include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
  • any number of selection systems are l ⁇ iown in the art that may be used to recover transformed cell lines.
  • Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al (1980) Cell 22:817-23) genes that can be employed in tk " or aprt* cells, respectively.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, ⁇ . et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression ofthe tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators ofthe polypeptides ofthe present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate 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 particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992) Prot. Exp. Purif.
  • the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • FACS fluorescence activated cell sorting
  • the polypeptide ofthe invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide ofthe first aspect ofthe invention or to regulate the activity of a polypeptide ofthe first aspect ofthe invention.
  • Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide ofthe invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation ofthe polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide ofthe present invention comprises:
  • a further preferred method for identifying an agonist or antagonist of a polypeptide ofthe invention comprises:
  • the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.
  • the method for identifying agonist or antagonist of a polypeptide ofthe present invention comprises: determining the inhibition of binding of a ligand to cells which have a polypeptide ofthe invention on the surface thereof, or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide.
  • a compound capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist.
  • the ligand is labelled. More particularly, a method of screening for a polypeptide antagonist or agonist compound comprises the steps of:
  • step (c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium; (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c); and
  • step (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
  • competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology ofthe manipulated cells. For example, such experiments reveal details of signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance (supplied by Biacore AB, Uppsala,
  • Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide ofthe invention discovered by the methods that are described above.
  • the invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
  • the pharmaceutical compositions should preferably comprise a therapeutically effective amount ofthe polypeptide, nucleic acid molecule, ligand, or compound ofthe invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targetted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • the pharmaceutical compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • an inhibitor compound as described above
  • a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • antagonists are antibodies.
  • such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • soluble forms ofthe polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question may be administered.
  • the polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • triplex DNA Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al (1994) In: Huber, B.E. and Bi. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY).
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules.
  • ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends ofthe molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone ofthe molecule.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
  • Gene therapy ofthe present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
  • in vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479.
  • a nucleic acid molecule encoding a polypeptide ofthe invention may be engineered for expression in a replication-defective retroviral vector.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera . pylori, and other pathogens.
  • vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood ofthe recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition ofthe sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • jet injection see, for example, www.powderject.com
  • jet injection may also be useful in the formulation of vaccine compositions.
  • This invention also relates to the use of nucleic acid molecules according to tlie present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules ofthe invention 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 variety of techniques.
  • Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et ah, Nature, 324, 163-166 (1986); Bej, et ah, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule ofthe invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels ofthe hybrid complex in the control sample is indicative of disease.
  • a further aspect ofthe invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and, c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included.
  • Deletions and insertions can be detected by a change in the size ofthe amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA ofthe invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion ofthe probe strand as an indication ofthe presence or absence of a disease-associated mutation in the corresponding portion ofthe DNA strand.
  • Such diagnostics are particularly useful for prenatal and even neonatal testing.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et ah, Genomics, 5, 874-879 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et ah, 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).
  • FISH Fluorescence in situ hybridization
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et ah, Science (1996) 274: 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et ah); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619).
  • Oligonucleotide pairs may range from two to over one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/25116 (Baldeschweiler et al).
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA.
  • Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays).
  • This aspect ofthe invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
  • Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide ofthe invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules known in the art may be used, several of which are described above.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit ofthe present invention may comprise:
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to diseases in which TNF- like proteins are implicated, particularly cell proliferative disorders, autoimmune/inflammatory disorders, genetic disorders, developmental disorders, nervous system disorders, metabolic disorders, infections and other pathological conditions; particularly immune disorders, such as autoimmune disease, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, and multiple sclerosis, inflammatory disorders, such as allergy, rhinitis, conjunctivitis, glomerulonephritis, uveitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, pancreatitis, digestive system inflammation, sepsis, endotoxic shock, septic shock, cachexia, myalgia, ankylosing spondylitis, myasthenia gravis, post-viral fatigue syndrome, pulmonary disease, respiratory distress syndrome, asthma, chronic-obstructive pulmonary disease, airway inflammation, wound healing,
  • Figure 1 Genome ThreaderTM alignment of BAB71417.1 and 1TNR:A
  • Figure 2 InterProScan results using BAB71417.1 (labelled INSP068) as a query sequence.
  • Figure 3 CDD results using BAB71417.1 (labelled INSP068) as a query sequence.
  • Figure 4 NCBI PSI-BLAST results using BAB71417.1 (labelled INSP068) as a query sequence.
  • Figure 5 Inpharmatica Genome ThreaderTM results of search using BAB71417.1.
  • Figure 6 Nucleotide sequence of INSP068 (SEQ ID NO:l) with translation.
  • Figure 7 Nucleotide sequence with translation of PCR product cloned from genomic DNA using primers INSP068-CP1 and INSP068-CP2.
  • Figure 8 Map of pCR4-TOPO-INSP068.
  • Figure 9 Map of Gateway entry vector pDONR201.
  • Figure 10 Map of pDONR201-INSP068-6HIS.
  • Figure 11 Map of expression vector pEAK12d.
  • Figure 12 Map of pEAK12d-_NSP068-6HIS.
  • Human TNF_beta In order to initiate a search for novel, distantly related TNF-like proteins, an archetypal TNF-like protein family member, Human TNF_beta is chosen. More specifically, the search is initiated using a structure from the Protein Data Bank (PDB) which is operated by the Research Collaboratory for Structural Bioinformatics. The structure chosen is Human TNF_beta, PDB code 1TNR:A. A search of the BiopendiumTM for homologues of 1TNR:A takes place and returns, amongst other known TNF-like protein members, a protein of apparently unknown function, BAB71417.1.
  • PDB Protein Data Bank
  • the Inpharmatica Genome ThreaderTM has identified residues 29-127 of a sequence, BAB71417.1, as having an equivalent structure to residues 34-144 of Human TNF beta (PDB code: 1TNR:A). Having a structure equivalent to 1TNR:A suggests that BAB71417.1 is a protein that functions as a TNF-like protein. The Inpharmatica Genome Threader identifies this with 63.4% confidence.
  • a structure-sequence alignment between the sequences of BAB71417.1 and 1TNR:A is carried out and shown in Figure 1 (the BAB71417.1 sequence is labelled INSP068 in the figure).
  • BAB71417.1 In order to view what is known in the public domain secondary databases about BAB71417.1, the InterPro database is queried with BAB71417.1 ( Figure 2). h terPro returns one hit to BAB71417.1, an ATP/GTP-binding site motif A (P-loop). This motif is not known to be associated with TNF molecules, and thus, noone would have been able to predict from the InterPro results that BAB71417.1 encodes a TNF-like protein.
  • CDD NCBI conserveed Domain Database
  • the sequence BAB71417.1 was also entered into the NCBI public domain PSI-Blast server. Querying NCBI PSI-Blast with BAB71417.1 through 3 positive iterations gives one significant hit - the hypothetical protein sequence XM_088653 ( Figure 4). XM_088653 is identical in sequence to BAB71417.1 and is not annotated by NCBI PSI-Blast as being a member ofthe TNF family.
  • BAB71417.1 is now used as the query sequence in the BiopendiumTM.
  • the Inpharmatica Genome ThreaderTM identifies 9 hits of which only the top hit is shown ( Figure 5).
  • the Inpharmatica Genome ThreaderTM ( Figure 5) identifies residues 29-127 of BAB71417.1 as having a stracture the same as Human TNF__beta (PDB code: 1TNR:A) with 63.4% confidence.
  • PDB code: 1TNR:A Human TNF__beta
  • the PCR was performed in a final volume of 50 ⁇ l containing IX AmpliTaqTM buffer, 200 ⁇ M dNTPs, 50 pmoles of each cloning primer, 2.5 units of AmpliTaqTM (Perkin Elmer) and 100 ng of genomic DNA (Novagen Inc.) using an MJ Research DNA Engine, programmed as follows: 94 °C, 2 min; 40 cycles of 94 °C, 1 min, 55 °C, 1 min, and 72 °C, 1 min; followed by 1 cycle at 72 °C for 7 min and a holding cycle at 4 °C.
  • PCR products were visualized on 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) and PCR products migrating at the predicted molecular mass (513 bp) were purified from the gel using the Wizard PCR Preps DNA Purification System (Promega). PCR products eluted in 50 ⁇ l of sterile water were either subcloned directly or stored at - 20 °C.
  • PCR primers having a length of between 18 and 25 bases were designed for amplifying the coding sequence of the virtual cDNA using Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). PCR primers were optimized to have a Tm close to 55 + 10 °C and a GC content of 40- 60%. Primers were selected which had high selectivity for the target sequence INSP068 (little or no none specific priming). 2.3 Subcloning of PCR Products
  • PCR products were subcloned into the topoisomerase I modified cloning vector (pCR4- TOPO) using the TA cloning kit purchased from the Invitrogen Corporation, using the conditions specified by the manufacturer. Briefly, 4 ⁇ l of gel purified PCR product was incubated for 15 min at room temperature with 1 ⁇ l of TOPO vector and 1 ⁇ l salt solution. The reaction mixture was then transformed into E. coli strain TOP 10 (Invitrogen) as follows: a 50 ⁇ l aliquot of One Shot TOP 10 cells was thawed on ice and 2 ⁇ l of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 °C for exactly 30 s.
  • E. coli strain TOP 10 Invitrogen
  • Colonies were inoculated into 50 ⁇ l sterile water using a sterile toothpick. A 10 ⁇ l aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 ⁇ l as described above, except the primers used were T7 and T3 (20 pmoles of each). The cycling conditions were as follows: 94 °C, 2 min; 30 cycles of 94 °C, 30 sec, 47 °C, 30 sec and 72 °C, 1 min. Samples were then maintained at 4 °C (holding cycle) before further analysis.
  • PCR reaction products were analyzed on 1 % agarose gel in 1 X TAE buffer. Colonies which gave approximately the expected PCR product size (513 bp cDNA + 105 bp due to the multiple cloning site or MCS) were grown up overnight at 37 °C in 5 ml L-Broth (LB) containing ampicillin (100 ⁇ g /ml), with shaking (220 rpm) at 37 °C.
  • Miniprep plasmid DNA was prepared from 5 ml cultures using a Qiaprep Turbo 9600 robotic system (Qiagen) or manually using a Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 100 ⁇ l of sterile water. The DNA concentration was measured using an Eppendorf BO photometer. Plasmid DNA (200-500 ng) was subjected to DNA sequencing with T7 and T3 primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 1. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • Example 3 Construction of a plasmid for the expression of INSP068 in HEK293/EBNA cells
  • the first stage ofthe Gateway cloning process involves a two step PCR reaction, which generates the ORF of INSP068 flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA).
  • 6HIS in frame 6 histidine
  • the first PCR reaction (in a final volume of 50 ⁇ l) contains: 1.5 ⁇ l of pCR4 TOPO- INSP068 (plasmid ID 13165), 1.5 ⁇ l dNTPs (10 mM), 5 ⁇ l of 10X Pfx polymerase buffer, 1 ⁇ l MgSO 4 (50 mM), 0.5 ⁇ l each of gene specific primer (100 ⁇ M) (INSP068-EX1 and INSP068-EX2) and 0.5 ⁇ l Platinum Pfx DNA polymerase (Invitrogen).
  • PCR reaction was performed using an initial denaturing step of 95°C for 1 min, followed by 10 cycles of 94 °C for 15 s; 55 °C for 30 s and 68°C for 1 min; and a holding cycle of 4 °C.
  • PCR products were purified directly from the reaction mixture using the Wizard PCR prep DNA purification system (Promega) according to the manufacturer's instructions.
  • the second PCR reaction (in a final volume of 50 ⁇ l) contained 10 ⁇ l purified PCR product, 1.5 ⁇ l dNTPs (10 mM), 5 ⁇ l of 10X Pfx polymerase buffer, 1 ⁇ l MgSO 4 (50 mM), 0.5 ⁇ l of each Gateway conversion primer (100 ⁇ M) (GCP forward and GCP reverse) and 0.5 ⁇ l of Platinum Pfx DNA polymerase.
  • the conditions for the 2nd PCR reaction were: 95 °C for 1 min; 4 cycles of 94 °C, 15 sec; 50 °C, 30 sec and 68 °C for 1 min; 25 cycles of 94 °C, 15 sec; 55 °C , 30 sec and 68 °C, 1 min; followed by a holding cycle of 4 °C.
  • PCR products (Gateway compatible INSP068 ORF) were purified using the Wizard PCR prep DNA purification system (Promega).
  • the second stage of the Gateway cloning process involves subcloning of the Gateway modified PCR product into the Gateway entry vector pDONR201 (Invitrogen, figure 9) as follows: 5 ⁇ l of purified PCR product is incubated with 1.5 ⁇ l pDONR201 vector (0.1 ⁇ g/ ⁇ l), 2 ⁇ l BP buffer and 1.5 ⁇ l of BP clonase enzyme mix (Invitrogen) at RT for 2 h. The reaction was stopped by addition of proteinase K (2 ⁇ g) and incubated at 37°C for a further 10 min. An aliquot of this reaction (2 ⁇ l) was added to 50 ul of E. coli TOP 10 competent cells (Invitrogen) and incubated on ice for 30 min.
  • the mixture was then heat shocked at 42°C for exactly 30 s, returned to ice for 2 min and then 250 ⁇ l of prewarmed (RT) SOC medium was added.
  • the transformation mix was incubated at 37° C for 1 hour with shaking at 220 rpm.
  • the transformation mixture (100 ⁇ l) was then plated on L-broth (LB) plates containing kanamycin (40 ⁇ g/ml) and incubated overnight at 37 °C.
  • LB L-broth
  • Six of the resultant kanamycin resistant colonies were inoculated into 5 ml of L-broth containing kanamycin (40 ⁇ g/ml) and incubated overnight at 37° C with shaking at 220 ⁇ m.
  • Mini-prep plasmid DNA was prepared from the 5 ml cutures using a Qiaprep Turbo 9600 robotic system (Qiagen). Plasmid DNA (200-500 ng) was subjected to DNA sequencing with pENTR-F and pENTR-R primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 1. Sequencing reactions were purified using Dye- Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • Plasmid eluate (1.5 ⁇ l) from one clone which gave the correct sequence (pDONR201-INSP068-6HIS, plasmid ID 13423) ( Figure 10) was then used in a recombination reaction containing 1.5 ⁇ l pEAK12d vector ( Figure 11) (0.1 ⁇ g / ⁇ l), 2 ⁇ l LR buffer and 1.5 ⁇ l of LR clonase (Invitrogen) in a final volume of 10 ⁇ l.
  • the mixture was incubated at RT for 1 h, stopped by addition of proteinase K (2 ⁇ g) and incubated at 37°C for a further 10 min. An aliquot of this reaction was used to transform E.
  • coli DH10B cells by electroporation as follows: a 20 ⁇ l aliquot of DH10B electrocompetent cells (Invitrogen) was thawed on ice and 1 ⁇ l of the LR reaction mix was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene-PulserTM according to the manufacturer's recommended protocol. SOC media (1 ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for 1 h at 37 °C.
  • Plasmid mini prep DNA was isolated from 4 independent ampicillin resistant clones using a Qiaprep Turbo 9600 robotic system (Qiagen) or manually using a Wizard Plus SV minipreps kit (Promega) and sequence verified as described above using the pEAK12d F and pEAK12d R primers.
  • a confrol reaction may be set up in which reverse transcription was not undertaken (a - RT control).
  • RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers.
  • ABSI Multiscript reverse transcriptase
  • a control reaction is set up in which all the constituents are added except the reverse transcriptase (-RT confrol).
  • PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls.
  • INSP068-specific primers may readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue.
  • Any suitable cDNA libraries may be used to screen for the INSP068 transcripts, not only those generated as described above.
  • tissue distribution pattern of the INSP068 polypeptides will provide further useful information in relation to the function of those polypeptides.
  • further experiments may now be performed using the pEAK12d-INSP068- 6HIS expression vectors. Transfection of mammalian cell lines with these vectors may enable the high level expression of the INSP068 proteins and thus enable the continued investigation of the functional characteristics of the INSP068 polypeptides.
  • the following material and methods are an example of those suitable in such experiments: 4.1.
  • Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogen) are maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH).
  • Ex-cell VPRO serum-free medium seed stock, maintenance medium, JRH.
  • cells are seeded in 2x T225 flasks (50 ml per flask in DMEM / F12 (1:1) containing 2% FBS seeding medium (JRH) at a density of 2x105 cells/ ml).
  • plasmid DNA is co-transfected with GFP (fluorescent reporter gene) DNA.
  • GFP fluorescent reporter gene
  • the transfection mix is then added to the 2xT225 flasks and incubated at 370C (5%CO2) for 6 days. Confirmation of positive transfection may be carried out by qualitative fluorescence examination at day 1 and day 6 (Axiovert 10 Zeiss).
  • Scale-up batches may be produced by following the protocol called "PEI transfection of suspension cells", referenced BP/PEI/HH/02/04, with PolyEthylenelmine from Polysciences as transfection agent.
  • the culture medium sample containing the recombinant protein with a C-terminal 6His tag is diluted with cold buffer A (50 mM NaH 2 PO 4 ; 600 mM NaCI; 8.7 % (w/v) glycerol, pH 7.5).
  • the sample is filtered then through a sterile filter (Millipore) and kept at 4°C in a sterile square media bottle (Nalgene).
  • the purification is performed at 4°C on the VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic).
  • the purification procedure is composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm, 0.83 ml), followed by gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column (1,0 x 10 cm).
  • the metal affinity column is regenerated with 30 column volumes of EDTA solution (100 mM EDTA; 1 M NaCI; pH 8.0), recharged with Ni ions through washing with 15 column volumes of a 100 mM NiSO solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (50 mM NaH 2 PO 4 ; 600 mM NaCI; 8.7 % (w/v) glycerol, 400 mM; imidazole, pH 7.5), and finally equilibrated with 15 column volumes of buffer A containing 15 mM imidazole.
  • EDTA solution 100 mM EDTA; 1 M NaCI; pH 8.0
  • the sample is transferred, by the Labomatic sample loader, into a 200 ml sample loop and subsequently charged onto the Ni metal affinity column at a flow rate of 10 ml/min.
  • the column is washed with 12 column volumes of buffer A, followed by 28 column volumes of buffer A containing 20 mM imidazole. During the 20 mM imidazole wash loosely attached contaminating proteins are eluted from the column.
  • the recombinant His-tagged protein is finally eluted with 10 column volumes of buffer B at a flow rate of 2 ml/min, and the eluted protein is collected.
  • the Sephadex G-25 gel-filtration column is regenerated with 2 ml of buffer D (1.137 M NaCI; 2.7 mM KC1; 1.5 mM KH 2 PO 4 ; 8 mM Na 2 HPO ; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137 mM NaCI; 2.7 mM KC1; 1.5 mM KH 2 PO 4 ; 8 mM Na 2 HPO 4 ; 20 % (w/v) glycerol; pH 7.4).
  • the peak fraction eluted from the Ni-column is automatically loaded onto the Sephadex G-25 column through the integrated sample loader on the VISION and the protein is eluted with buffer C at a flow rate of 2 ml/min.
  • the fraction was filtered through a sterile centrifugation filter (Millipore), frozen and stored at -80°C.
  • An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) Western blot with anti-His antibodies.
  • the NuPAGE gel may be stained in a 0.1 % Coomassie blue R250 staining solution (30 % methanol, 10 % acetic acid) at room temperature for 1 h and subsequently destained in 20 % methanol, 7.5 % acetic acid until the background is clear and the protein bands clearly visible.
  • the proteins are elecfrofransferred from the gel to a nitrocellulose membrane.
  • the membrane is blocked with 5 % milk powder in buffer E (137 mM NaCI; 2.7 mM KC1; 1.5 mM KH 2 PO 4 ; 8 mM Na 2 HPO 4 ; 0.1 % Tween 20, pH 7.4) for 1 h at room temperature, and subsequently incubated with a mixture of 2 rabbit polyclonal anti-His antibodies (G-18 and H-15, 0.2ug/ml each; Santa Cruz) in 2.5 % milk powder in buffer E overnight at 4°C.
  • the membrane After a further 1 hour incubation at room temperature, the membrane is washed with buffer E (3 x 10 min), and then incubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5 % milk powder for 2 hours at room temperature. After washing with buffer E (3 x 10 minutes), the membrane is developed with the ECL kit (Amersham Pharmacia) for 1 min. The membrane is subsequently exposed to a Hyperfilm (Amersham Pharmacia), the film developed and the western blot image visually analysed.
  • DAKO secondary HRP-conjugated anti-rabbit antibody
  • the protein concentration may be determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.
  • BCA protein assay kit Pieris

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Abstract

L'invention porte sur une protéine BAB71417.1 identifiée ci-présent en tant que nouvel élément de la famille de type TNF (facteur de nécrose des tumeurs) des cytokines, et sur l'utilisation de cette protéine et de la séquence d'acide nucléique à partir du gène de codage dans le diagnostic, la prévention et le traitement de maladies.
PCT/GB2003/002510 2002-06-11 2003-06-11 Proteine de type tnf WO2003104278A1 (fr)

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GBGB0213355.1A GB0213355D0 (en) 2002-06-11 2002-06-11 Protein
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1293569A2 (fr) * 2001-09-14 2003-03-19 Helix Research Institute Clones d'ADNc complets

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1293569A2 (fr) * 2001-09-14 2003-03-19 Helix Research Institute Clones d'ADNc complets

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 31 August 2001 (2001-08-31), ISHIBASHI, T ET AL.: "Homo sapiens cDNA FLJ32742 fis, clone TESTI2001352", XP002255991, Database accession no. AK057304 *
INPHARMATICA: "Biopendium TM", XP002255990 *
JONES D T: "GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences.", JOURNAL OF MOLECULAR BIOLOGY. ENGLAND 9 APR 1999, vol. 287, no. 4, 9 April 1999 (1999-04-09), pages 797 - 815, XP004455936, ISSN: 0022-2836 *

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