WO2008003925A2 - Protéine du complément - Google Patents

Protéine du complément Download PDF

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Publication number
WO2008003925A2
WO2008003925A2 PCT/GB2007/002334 GB2007002334W WO2008003925A2 WO 2008003925 A2 WO2008003925 A2 WO 2008003925A2 GB 2007002334 W GB2007002334 W GB 2007002334W WO 2008003925 A2 WO2008003925 A2 WO 2008003925A2
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Prior art keywords
seq
polypeptide
disease
nucleic acid
acid molecule
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PCT/GB2007/002334
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English (en)
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WO2008003925A3 (fr
Inventor
Richard Joseph Fagan
Stephen Noel Fitzgerald
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Ares Trading S.A.
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Publication of WO2008003925A2 publication Critical patent/WO2008003925A2/fr
Publication of WO2008003925A3 publication Critical patent/WO2008003925A3/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a

Definitions

  • This invention relates to a novel protein, termed INSP191, herein identified as a secreted protein, in particular, as a complement protein and to the use of this protein and nucleic acid sequence from the encoding gene in the diagnosis, prevention and treatment of disease.
  • Secreted Proteins The ability for cells to make and secrete extracellular proteins is central to many biological processes. Enzymes, growth factors, extracellular matrix proteins and signalling molecules are all secreted by cells. This is through fusion of a secretory vesicle with the plasma membrane. In most cases, but not all, proteins are directed to the endoplasmic reticulum and into secretory vesicles by a signal peptide. Signal peptides are cis-acting sequences that affect the transport of polypeptide chains from the cytoplasm to a membrane bound compartment such as a secretory vesicle. Polypeptides that are targeted to the secretory vesicles are either secreted into the extracellular matrix or are retained in the plasma membrane.
  • polypeptides that are retained in the plasma membrane will have one or more transmembrane domains.
  • secreted proteins that play a central role in the functioning of a cell are cytokines, hormones, extracellular matrix proteins (adhesion molecules), proteases, and growth and differentiation factors. Description of some of the properties of these proteins follows later in the specification.
  • complement proteins are highly likely to be implicated in disease. Identification of such proteins will also be of importance in increasing the understanding of the underlying pathways that lead to the diseases states and associated disease states, mentioned above, and in developing more effective gene and/or drug therapies to treat these disorders.
  • the invention is based on the discovery that the INSP 191 polypeptide is a complement protein.
  • polypeptide which:
  • (i) comprises the amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:
  • (ii) is a fragment thereof which is a complement protein, or having an antigenic determinant in common with the polypeptides of (i); or (iii) is a functional equivalent of (i) or (ii).
  • the polypeptide according to this first aspect of the invention comprises the amino acid sequence as recited in SEQ ID NO:84 or SEQ ID NO:88.
  • polypeptide which consists of the amino acid sequence as in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:
  • polypeptide having the sequence recited in SEQ ID NO: 2 is referred to hereafter as "INSPl 91 exon 1 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 4 is referred to hereafter as "INSPl 91 exon 2 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 6 is referred to hereafter as "INSP191 exon 3 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 8 is referred to hereafter as "INSP 191 exon 4 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 10 is referred to hereafter as "INSPl 91 exon 5 polypeptide".
  • polypeptide having the sequence recited in SEQ ID NO: 12 is referred to hereafter as "INSP 191 exon 6 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 14 is referred to hereafter as "INSP 191 exon 7 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 16 is referred to hereafter as "INSP 191 exon 8 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as "INSP 191 exon 9 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 20 is referred to hereafter as "INSP191 exon 10 polypeptide”.
  • polypeptide having the sequence recited in SEQ ID NO: 22 is referred to hereafter as "INSP 191 exon 11 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 24 is referred to hereafter as "INSP 191 exon 12 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 26 is referred to hereafter as "INSPl 91 exon 13 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 28 is referred to hereafter as "INSP 191 exon 14 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 30 is referred to hereafter as "INSP191 exon 15 polypeptide”.
  • polypeptide having the sequence recited in SEQ ID NO: 32 is referred to hereafter as "INSP191 exon 16 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 34 is referred to hereafter as "INSP 191 exon 17 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 36 is referred to hereafter as "INSP191 exon 18 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 38 is referred to hereafter as "INSPl 91 exon 19 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 40 is referred to hereafter as "INSP191 exon 20 polypeptide".
  • polypeptide having the sequence recited in SEQ ID NO: 42 is referred to hereafter as "INSPl 91 exon 21 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 44 is referred to hereafter as "INSPl 91 exon 22 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 46 is referred to hereafter as "INSP 191 exon 23 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 48 is referred to hereafter as "INSP 191 exon 24 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 50 is referred to hereafter as "INSPl 91 exon 25 polypeptide”.
  • polypeptide having the sequence recited in SEQ ID NO: 52 is referred to hereafter as "INSPl 91 exon 26 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 54 is referred to hereafter as "INSP 191 exon 27 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 56 is referred to hereafter as "INSP191 exon 28 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 58 is referred to hereafter as "INSP 191 exon 29 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 60 is referred to hereafter as "INSP191 exon 30 polypeptide”.
  • polypeptide having the sequence recited in SEQ ID NO: 62 is referred to hereafter as "INSPl 91 exon 31 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 64 is referred to hereafter as "INSP191 exon 32 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 66 is referred to hereafter as "INSP 191 exon 33 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 68 is referred to hereafter as "INSP 191 exon 34 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 70 is referred to hereafter as "INSP191 exon 35 polypeptide".
  • polypeptide having the sequence recited in SEQ ID NO: 72 is referred to hereafter as "INSPl 91 exon 36 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 74 is referred to hereafter as "INSP191 exon 37 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 76 is referred to hereafter as "INSP 191 exon 38 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 78 is referred to hereafter as "INSP191 exon 39 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 80 is referred to hereafter as "INSPl 91 exon 40 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 82 is referred to hereafter as "INSP 191 exon 41 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 84 is referred to hereafter as "INSPl 91 polypeptide”.
  • the Applicant does not wish to be bound by this theory, it is postulated that the first 21 amino acids of the INSP 191 polypeptide form a signal peptide.
  • the INSP 191 exon 1 polypeptide without this postulated signal sequence is recited in SEQ ID NO: 86.
  • the full length INSPl 91 polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO: 88.
  • the polypeptide having the sequence recited in SEQ ID NO: 86 is referred to hereafter as "the INSPl 91 exon 1 mature polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 88 is referred to hereafter as "the INSPl 91 mature polypeptide".
  • INSPl 91 polypeptides includes the INSPl 91 exon 1 polypeptide, the INSP 191 exon 2 polypeptide, the INSP 191 exon 3, the INSP 191 exon 4 polypeptide, the INSP 191 exon 5 polypeptide, the INSPl 91 exon 6 polypeptide, the INSPl 91 exon 7 polypeptide, the INSP 191 exon 8 polypeptide, the INSP191 exon 9 polypeptide, the INSP 191 exon 10 polypeptide, the INSPl 91 exon 11 polypeptide, the INSPl 91 exon 12 polypeptide, the INSPl 91 exon 13 polypeptide, the INSP 191 exon 14 polypeptide, the INSP 191 exon 15 polypeptide, the INSPl 91 exon 16 polypeptide, the INSP191 exon 17 polypeptide, the INSP191 exon 18 polypeptide, the INSP 191 exon 19 polypeptide, the INSP 191 exon 19 polypeptide, the
  • complement protein refers to proteins that play a role in the complement system of innate immunity.
  • a polypeptide according to any one of the above-described aspects of the invention functions as a complement protein.
  • functions as a complement protein we refer to polypeptides that comprise amino acid sequence or structural features that can be identified as conserved features within the polypeptides of the complement proteins. Such function may be assayed using techniques well known in the art such as the assay for total complement hemolytic activity reported by
  • the INSP 191 sequence is shown herein to contain alpha-2-macroglobulin, netrin and anaphylatoxin domains. This is characteristic of the complement proteins C3, C4 and
  • Complement is a major effector system of innate immunity (Morgan & Harris, 2003, MoI. Immunol. 40:159-170).
  • the complement system is a group of about 25 soluble and cell- surface proteins which interact to recognise, opsonise and clear or kill invading bacteria and viruses possessing a lipoprotein envelope as well as altered host cells (e.g. apoptotic or necrotic cells).
  • the soluble proteins make up 5% of the total protein content of human blood plasma.
  • the system promotes clearance by phagocytosis by attaching to targets which then interact with cells of the adaptive immune system, influencing the host response to microorganisms and potential autoantigens.
  • the complement system recognises targets by weak interactions between complement recognition proteins and charge or carbohydrate arrays on the target surfaces.
  • the system has a built in redundancy, being activated by three different pathways, called the classical pathway, the lectin pathway and the alternative pathway.
  • the alternative pathway is activated by invading micro-organisms and does not require antibody.
  • the six proteins C3, B, D, H, I and P together perform the functions of initiation, recognition and activation of this pathway which results in the formation of activator- bound C3/C5 convertase.
  • the classical pathway functions to mediate the specific antibody response. It is as elaborately controlled as the alternative pathway, although it does lack the spontaneous initiation ability (the antibody independent recognition function) and the feedback amplification mechanism.
  • Both activation pathways contain an initial enzyme that catalyses the formation of the target cell bound C3 convertase which, in turn, generates the C5 convertase. This results in the cleavage and activation of C5 and, therefore, in the assembly of the membrane attack complex (MAC).
  • MAC membrane attack complex
  • the MAC is assembled from five hydrophilic precursor proteins: C5, C6, C7, C8, and C9. Activation of the MAC is a consequence of the activity of either the classical or the alternative pathway on the surface of a cell. Through its metastable membrane binding site, the forming MAC binds firmly to target membranes owing to hydrophobic interactions with the lipid bilayer. The final events are the unfolding, the oligomerisation, or the polymerisation of C9, which causes the weakening of membrane structure, and the formation of transmembrane channels thus leading to osmotic lysis of the cell.
  • proinflammatory mediators C3a and C5a are essential for liver regeneration (see, for example, Strey CW, Markiewski M, Mastellos D, Vietnamesean R, Spruce LA, Greenbaum LE, Lambris JD. J Exp Med. 2003 Sep 15;198(6):913-23). Accordingly, this protein may have a useful function in counteracting the effects of toxic liver disease, a worldwide health problem in humans for which pharmacological treatments have yet to be discovered. For example, active chronic hepatitis leading to liver cirrhosis is a disease state, in which liver parenchymal cells are progressively destroyed by activated T cells.
  • ConA-induced liver toxicity is one of three experimental models of T-cell dependent apoptotic and necrotic liver injury described in mice.
  • Gal N D-Galactosamine sensitized mice challenged with either activating anti-CD3 monoclonal AB or with superantigen SEB develop severe apoptotic and secondary necrotic liver injury (Kusters S, Gastroenterology. 1996 Aug;l l l(2):462-71).
  • Injection of the T-cell mitogenic plant lectin ConA to non- sensitized mice results also in hepatic apoptosis that preceeds necrosis.
  • ConA induces the release of systemic TNF ⁇ and IFN ⁇ and various other cytokines. Both TNF ⁇ and IFN ⁇ are critical mediators of liver injury. Transaminase release 8 hours after the insult indicates severe liver destruction.
  • liver damage Several cell types have been shown to be involved in liver damage, including CD4 T cells, macrophages and natural killer cells (Kaneko J Exp Med 2000, 191, 105-114).
  • Anti-CD4 antibodies block activation of T cells and consequently liver damage (Tiegs et al. 1992, J Clin Invest 90, 196-203).
  • Pre-treatment of mice with monoclonal antibodies against CD8 failed to protect, whereas deletion of macrophages prevented the induction of hepatitis.
  • TNF ⁇ for example is one of the first cytokines produced after ConA injection and anti-TNF ⁇ antibodies confer protection against disease (Seino et al. 2001, Annals of surgery 234, 681). IFN ⁇ appears also to be a critical mediator of liver injury, since anti-IFN ⁇ antiserum significantly protect mice, as measured by decreased levels of transaminases in the blood of ConA- treated animals (see Kusters et al., above).
  • IFN ⁇ In liver injury, increased production of IFN ⁇ has been observed in patients with autoimmune or viral hepatitis.
  • transgenic mice expressing IFN ⁇ in the liver develop liver injury resembling chronic active hepatitis (Toyonaga et al. 1994, PNAS 91, 614-618).
  • IFN ⁇ may also be cytotoxic to hepatocytes, since in vitro IFN ⁇ induces cell death in mouse hepatocytes that was accelerated by TNF (Morita et al. 1995, Hepatology 21, 1585-1593).
  • Alpha-2-macroglobulin has been implicated in Alzheimer disease based on its ability to mediate the clearance and degradation of A-beta, the major component of amyloid beta deposits.
  • the sibship disequilibrium test SDT also revealed a significant association between A2M and AD.
  • polypeptides of the present invention may modulate a variety of physiological and pathological processes or disorders.
  • the biological activity or function of these polypeptides can be examined in systems that allow the study of such modulatory activities, using a variety of suitable assays.
  • the biological activity of INSPl 91 can be confirmed in at least one of the following assays: a. INSP191 can reduce infection, for example of Neisseria species, or b. INSP191 can modulate hepatocytes' regeneration or show activity in the mouse model of fulminant hepatitis as described in Example 5, or c. INSPl 91 can prevent blood clotting or platelet aggregation, or d. INSPl 91 can act on the dilatation or constriction of blood vessels, or e. INSP 191 can modulate blood pressure or blood coagulation, or f. INSP 191 can modulate fluid retention, or g. INSP 191 can modulate opsonization, or h. INSPl 91 can modulate complement activation.
  • INSP191 polypeptides The biological activity of INSP191 polypeptides can be demonstrated in mouse models of infectious diseases as described in Cluff et al. (Infect Immun. 2005 May;73(5):3044-52), Ahmed et al. (Lancet Infect Dis. 2004 Sep;4(9):566-74.), in Schriewer et al. (Methods MoI Biol. 2004;269:289-308.), in Medina (Drug Discovery Today: Disease Models; Vol. 1, No. 1, 2004, pp. 65-71), in Pincus (Drug Discovery Today: Disease Models; Vol. 1, No. 1, 2004, pp. 49-56), in de Oliveira et al. (Drug Discovery Today: Disease Models; Vol. 1, No. 1, 2004, pp.
  • INSP191 polypeptides can be demonstrated in mouse models of glomerulonephritis as described in Kikuchi et al. (J Immunol. 2002 Oct 15; 169(8) :4644- 50.), in Nordstrand et al. (Infect Immun. 1998 Jan;66(l):315-21.), or in Salama (Drug Discovery Today: Disease Models; Vol. 1, No. 4, 2004, pp. 457-463).
  • the polypeptides of the first aspect of the invention may further comprise a histidine tag.
  • the histidine tag is found at the C-terminal of the polypeptide.
  • the histidine tag comprises 1-10 histidine residues (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably the histidine tag comprises 6 histidine residues.
  • an “antigenic determinant” of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR).
  • an "antigenic determinant” may be a site on the surface of a polypeptide of the present invention to which a single antibody molecule binds.
  • an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities.
  • the antibody is immunospecific to a polypeptide of the invention.
  • the antibody is immunospecific to a polypeptide of the invention, which is not part of a fusion protein.
  • the antibody is immunospecific to INSP191 or a fragment thereof.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the "antigenic determinant” refers to a particular chemical group on a polypeptide of the present invention that is antigenic, i.e. that elicit a specific immune response.
  • the invention provides a purified nucleic acid molecule which encodes a polypeptide of the first aspect of the invention.
  • purified nucleic acid molecule preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
  • genomic DNA are specifically excluded from the scope of the invention.
  • genomic DNA larger than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp are specifically excluded from the scope of the invention.
  • the "purified nucleic acid molecule" consists of cDNA only.
  • the purified nucleic acid molecule comprises the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSPl 91 exon 1 polypeptide), SEQ ID NO: 3 (encoding the INSP191 exon 2 polypeptide), SEQ ID NO:5 (encoding the INSP191 exon 3 polypeptide), SEQ ID NO:7 (encoding the INSPl 91 exon 4 polypeptide), SEQ ID NO:9 (encoding the INSP 191 exon 5 polypeptide), SEQ ID NO:11 (encoding the INSP191 exon 6 polypeptide), SEQ ID NO: 13 (encoding the INSPl 91 exon 7 polypeptide), SEQ ID NO: 15 (encoding the INSP 191 exon 8 polypeptide), SEQ ID NO: 17 (encoding the INSPl 91 exon 9 polypeptide), SEQ ID NO:19 (encoding the INSP191 exon 10 polypeptide), SEQ ID NO:21 (encoding the INSP191 exon
  • the invention further provides that the purified nucleic acid molecule consists of the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP 191 exon 1 polypeptide), SEQ ID NO:3 (encoding the INSPl 91 exon 2 polypeptide), SEQ ID NO:5 (encoding the INSPl 91 exon 3 polypeptide), SEQ ID NO:7 (encoding the INSP191 exon 4 polypeptide), SEQ ID NO:9 (encoding the INSP191 exon 5 polypeptide), SEQ ID NO.l l (encoding the INSPl 91 exon 6 polypeptide), SEQ ID NO: 13 (encoding the INSP 191 exon 7 polypeptide), SEQ ID NO: 15 (encoding the INSP 191 exon 8 polypeptide), SEQ ID NO: 17 (encoding the INSP 191 exon 9 polypeptide), SEQ ID NO: 19 (encoding the INSP 191 exon 10 polypeptide), SEQ ID NO:21 (encoding the IN
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM 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.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the invention provides a ligand which binds specifically to complement proteins of the first aspect of the invention.
  • the ligand inhibits the function of a polypeptide of the first aspect of the invention which is a complement protein.
  • Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the 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 of the first aspect of the invention.
  • Such compounds may be identified using the assays and screening methods disclosed herein.
  • 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 of the function of the INSP191 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease.
  • Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.
  • Another aspect of this invention resides in the use of an INSP 191 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against complement related disorders.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of complement related disorders, comprising determining the ability of a compound to bind to an INSP 191 gene or polypeptide, or a fragment thereof.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of complement related disorders, comprising testing for modulation of the activity of an INSP 191 gene or polypeptide, or a fragment thereof.
  • 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 diseases in which complement proteins are implicated.
  • the disease is selected from infectious disease, glomerular disease, cardiovascular disease or hematology-related disorder or other disorders.
  • the infectious disease is selected from Systemic Fungal Disease, Rickettsial Disease, Chlamydial Disease, Parasitic Infection, viral Disease, Abscess, Human Immunodeficiency Virus Infection, Bacteremia, Septic Shock, sexually Transmitted Disease or Bacterial Disease.
  • the bacterial disease is selected from a disease caused by Gram-Positive Cocci, caused by Gram-Negative Aerobic Cocci, caused by Gram-Positive Bacilli, caused by Gram-Negative Bacilli, caused by Anaerobic Bacilli, caused by Spirochetes or caused by Mycobacteria.
  • a "Gram-Negative Aerobic Cocci” refers herein to an organism of the genus Neisseria including N. Meningitidis, N. Gonorrhoeae, and numerous saprophytic Neisseria sp. that commonly inhabit the oropharynx, vagina, or colon.
  • the disease caused by Gram-Negative Aerobic Cocci is selected from meningitis, bacteremia, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrheal infection, acute cacterial meningitis or Meningococcal infection.
  • Bacteremia herein refers to bacteria in the bloodstream.
  • Septic shock herein refers to sepsis with hypoperfusion and hypotension refractory to fluid therapy.
  • Sepsis or “systemic inflammatory response syndrome” herein refers to a serious infection, localized or bacteremic, that is accompanied by systemic manifestations of inflammation. Sepsis due to bacteremia herein refers to septicemia.
  • the Parasitic Infections is selected from Extraintestinal Protozoa infection, infection with Free-Living Amebas, Intestinal Protozoa infection, Nematode (Roundworm) Infection, Trematode (Fluke) infection or Cestodes (Tapeworms) infection.
  • the Extraintestinal Protozoa infection is malaria.
  • the viral disease is selected from Respiratory Viral Disease, Herpesvirus Infection, Central Nervous System Viral Disease, Arbovirus or Arenavirus Disease.
  • the glomerular disease is selected from nephritic syndrome, nephrotic syndrome, primary glomerular disease, or secondary renal disease.
  • the primary glomerular disease is selected from minimal change disease, focal segmental glomerulosclerosis, membranous glomerulonelhritis, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, IgA nephropathy, rapidly progressive glomerulonephritis, or fibrillary glomerulonephritis.
  • the nephritic syndrome is selected from hematuria, hypertension, renal insufficiency, edema, acute glomerulonephritis, transient glomerulonephritis, postinfectious glomerulonephritis, fulminant glomerulonephritis, rapidly progressive glomerulonephritis (RPGN), indolent Glomerulonephritis, IgA nephropathy, Crescentic glomerulonephritis, Pauci-immune RPGN, Immune complex RPGN, Anti-GBM antibody disease autoimmunity, primary renal hematuric-proteinuric syndrome, asymptomatic hematuric-proteinuric syndrome, chronic nephritic-proteinuric syndrome, chronic glomerulonephritis, slowly progressive glomerular disease.
  • RPGN rapidly progressive glomerulonephritis
  • the cardiovascular disorder is selected from Cardiac and Respiratory Arrest, Valvular Heart Disease, Arterial Hypertension, Endocarditis, Orthostatic Hypotension, Syncope, Pericardial Disease, Arteriosclerosis, Cardiac Tumor, Coronary Artery Disease, Disease of the Aorta and Its Branches, Heart Failure, Peripheral Vascular Disorder, Shock, Athletic Heart Syndrome or Arrhythmia.
  • the hematology-related disorder is selected from Anemia, Histiocytic Syndrome, Iron Overload related disorder, Leukemia, Lymphoma, Myeloproliferative Disorder, Plasma Cell Dyscrasia, Hemostasis and Coagulation Disorder, Disorder of the Spleen, Thrombotic Disorder, Platelet Disorder, Vascular Bleeding Disorder, Leukopenia, Lymphocytopenia or AID S -Associated Hematologic Disorder and Malignancy.
  • These molecules may also be used in the manufacture of a medicament for the treatment such diseases.
  • moieties of the present invention i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention
  • 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.
  • 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, of the 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 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.
  • the invention provides for the use of a polypeptide of the first aspect of the invention as a complement protein, such as in the immune response, particularly against pathogens.
  • 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 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, in conjunction with a pharmaceutically- acceptable carrier.
  • the present 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 the manufacture of a medicament for the diagnosis or treatment of a disease.
  • the disease is selected from infectious disease, glomerular disease, cardiovascular disease or hematology-related disorder or other disorder.
  • the infectious disease is selected from Systemic Fungal Disease, Rickettsial Disease, Chlamydial Disease, Parasitic Infection, Viral Disease, Abscess, Human Immunodeficiency Virus Infection, Bacteremia, Septic Shock, sexually Transmitted Disease or Bacterial Disease.
  • the bacterial disease is selected from a disease caused by Gram-Positive Cocci, caused by Gram-Negative Aerobic Cocci, caused by Gram-Positive Bacilli, caused by Gram-Negative Bacilli, caused by Anaerobic Bacilli, caused by Spirochetes or caused by Mycobacteria.
  • the disease caused by Gram-Negative Aerobic Cocci is selected from meningitis, bacteremia, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrheal infection, acute cacterial meningitis or Meningococcal infection.
  • the Parasitic Infections is selected from Extraintestinal Protozoa infection, infection with Free-Living Amebas, Intestinal Protozoa infection, Nematode (Roundworm) Infection, Trematode (Fluke) infection or Cestodes (Tapeworms) infection.
  • the Extraintestinal Protozoa infection is malaria.
  • the viral disease is selected from Respiratory Viral Disease, Herpesvirus Infection, Central Nervous System Viral Disease, Arbovirus or Arenavirus Disease.
  • the glomerular disease is selected from nephritic syndrome, nephrotic syndrome, primary glomerular disease, or secondary renal disease.
  • the primary glomerular disease is selected from minimal change disease, focal segmental glomerulosclerosis, membranous glomerulonelhritis, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, IgA nephropathy, rapidly progressive glomerulonephritis, or fibrillary glomerulonephritis.
  • the nephritic syndrome is selected from hematuria, hypertension, renal insufficiency, edema, acute glomerulonephritis, transient glomerulonephritis, postinfectious glomerulonephritis, fulminant glomerulonephritis, rapidly progressive glomerulonephritis (RPGN), indolent Glomerulonephritis, IgA nephropathy, Crescentic glomerulonephritis, Pauci-immune RPGN, Immune complex RPGN, Anti-GBM antibody disease autoimmunity, primary renal hematuric-proteinuric syndrome, asymptomatic hematuric-proteinuric syndrome, chronic nephritic-proteinuric syndrome, chronic glomerulonephritis, slowly progressive glomerular disease.
  • RPGN rapidly progressive glomerulonephritis
  • the cardiovascular disorder is selected from Cardiac and Respiratory Arrest, Valvular Heart Disease, Arterial Hypertension, Endocarditis, Orthostatic Hypotension, Syncope, Pericardial Disease, Arteriosclerosis, Cardiac Tumor, Coronary Artery Disease, Disease of the Aorta and Its Branches, Heart Failure, Peripheral Vascular Disorder, Shock, Athletic Heart Syndrome or Arrhythmia.
  • the hematology-related disorder is selected from Anemia, Histiocytic Syndrome, Iron Overload related disorder, Leukemia, Lymphoma, Myeloproliferative Disorder, Plasma Cell Dyscrasia, Hemostasis and Coagulation Disorder, Disorder of the Spleen, Thrombotic Disorder, Platelet Disorder, Vascular Bleeding Disorder, Leukopenia, Lymphocytopenia or AIDS-Associated Hematologic Disorder and Malignancy.
  • the other disorder is selected from Alzheimers, systemic lupus erythematosus, multiple sclerosis or liver cancer, in particular hepatocellular carcinoma.
  • the invention provides a method of treating a disease in a patient comprising administering to the patient 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.
  • the polypeptide, nucleic acid molecule, 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.
  • antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
  • the INSPl 91 polypeptides are complement proteins and thus have roles in many disease states. Antagonists of the INSP 191 polypeptides are of particular interest as they provide a way of modulating these disease states.
  • 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 used in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • “functional equivalent” refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention.
  • a functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional equivalent” is intended to include the fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.
  • the "functional equivalent” may be a protein or nucleic acid molecule that exhibits any one or more of the functional activities of the polypeptides of the present invention.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays substantially similar activity compared with INSP191 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays substantially similar activity compared with INSP191 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • “functional equivalent” may be a protein or nucleic acid molecule that displays identical or higher activity compared with INSPl 91 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the “functional equivalent” may be a protein or nucleic acid molecule that displays 50%, 60%, 70%, 80%, 90%, 95%, 98%,
  • the "functional equivalent” may be a protein or polypeptide capable of exhibiting a substantially similar in vivo or in vitro activity as the polypeptides of the invention.
  • the "functional equivalent” may be a protein or polypeptide capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the polypeptides of the invention would.
  • a "functional equivalent” would be able, in an immunoassay, to diminish the binding of an antibody to the corresponding peptide (i.e., the peptide the amino acid sequence of which was modified to achieve the "functional equivalent") of the polypeptide of the invention, or to the polypeptide of the invention itself, where the antibody was raised against the corresponding peptide of the polypeptide of the invention.
  • An equimolar concentration of the functional equivalent will diminish the aforesaid binding of the corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50%, and most preferably between about 40% and 50%.
  • 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 of the 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 of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • a fusion protein may contain one or more 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).
  • a polypeptide of the invention that may comprise a sequence having at least 85% of homology with INSP191
  • fusion protein can be obtained by cloning a polynucleotide encoding a polypeptide comprising a sequence having at least 85% of homology with INSP191 in frame to the coding sequences for a heterologous protein sequence.
  • heterologous when used herein, is intended to designate any polypeptide other than a human INSPl 91 polypeptide.
  • heterologous sequences that can be comprised in the fusion proteins either at the N- or C-terminus, include: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc regions), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, and sequences allowing purification by affinity chromatography.
  • heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, 2003, Appl Microbiol Biotechnol, 60:523-33).
  • additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by the a stretch of Histidines forming the so-called "histidine tag" (Gentz et al.
  • the heterologous sequence can be eliminated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease.
  • the INSP 191 polypeptide may be purified by means of a hexa-histidine peptide fused at the C-terminus of INSP 191.
  • the fusion protein comprises an immunoglobulin region
  • the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length.
  • Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence comprising GIu- Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO: 89) introduced between the sequence of the substances of the invention and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids ⁇ i.e. an increased half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • the protein is fused to the constant region of an Ig molecule.
  • it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgGl, for example.
  • Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomelic or multimeric, hetero- or homomultimeric.
  • the functional derivative comprises at least one moiety attached to one or more functional groups, which occur as one or more side chains on the amino acid residues.
  • the moiety is a polyethylene (PEG) moiety. PEGylation may be carried out by known methods, such as the ones described in WO99/55377, for example.
  • 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 of the 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 of the present invention.
  • the 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 INSPl 91 polypeptides.
  • 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.
  • 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) of the INSP191 polypeptides.
  • 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.
  • Such substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; 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 of the amino acid residues includes a substituent group.
  • any substitution should be preferably a "conservative” or “safe” substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties (e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.
  • the literature provide many models on which the selection of conservative amino acids substitutions can be performed on the basis of statistical and physico-chemical studies on the sequence and/or the structure of proteins (Rogov SI and Nekrasov AN, 2001).
  • non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the complement protein may increase its ability to be reused and recycled, potentially increasing its therapeutic potency (Robinson CR, 2002). Immunogenic epitopes eventually present in the polypeptides of the invention can be exploited for developing vaccines (Stevanovic S,
  • amino acids derivatives included in peptide mimetics are those defined in Table 2.
  • a non-exhaustive list of amino acid derivatives also include aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydro- isoquinoline-3-COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidine-4- carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine, and phenylglycine.
  • amino acid derivative is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids.
  • the amino acid derivative may contain substituted or non-substituted, linear, branched, or cyclic alkyl moieties, and may include one or more heteroatoms.
  • the amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem- Novabiochem AG, Switzerland; Bachem, USA).
  • polypeptides of the first aspect of the invention typically have a degree of sequence identity with the INSP191 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.
  • 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 Threader technology that forms one aspect of the search tools used to generate the BiopendiumTM search database may be used (see PCT application WO 01/69507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP 191 polypeptides, are predicted to be complement proteins, by virtue of sharing significant structural homology with the INSP 191 polypeptide sequence.
  • significant structural homology is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.
  • Polypeptides may be divided into fragments and similarly fragments of functional equivalents may exist. Such fragments are identified by being members of the same protein family as the full-length polypeptide, or having an antigenic determinant in common with the full-length polypeptides.
  • 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 a polypeptide or one of the functional equivalents of that polypeptide.
  • 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.
  • Nucleic acid fragments according to the invention are preferably 10-5000 nucleotides in length, preferably 50-2500 nucleotides, preferably 100-1000 nucleotides, preferably 200- 500 nucleotides in length.
  • Polypeptide fragments according to the invention are preferably 10-1600 amino acids in length, preferably 50-1200, preferably 100-800, preferably 200- 400 amino acids in length.
  • Fragments of full length polypeptides may consist of combinations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or all 41 neighbouring exon sequences in the polypeptide sequences, respectively.
  • such combinations include exons 1 and 2, exons 2 and 3, exons 1 and 3 or exons 1, 2 and 3, and so on.
  • Such fragments are included in the present invention.
  • Fragments preferably contain an alpha-2-macroglobulin domain, for example, see those identified in Figure 3. Such fragments may be "free-standing", i.e.
  • the fragment of the 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 of the 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 other 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 of the 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 secreted proteins.
  • the affinity is at least 1.5-fold, 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 secreted proteins such as complement proteins.
  • a selected mammal such as a mouse, rabbit, goat or horse
  • a polypeptide of the first aspect of the invention 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.
  • the polypeptide can be conjugated to a carrier protein.
  • Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
  • 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 of the first aspect of the 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,
  • 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. USA, 88, 34181 (1991); and Hodgson et al, Bio/Technology, 9, 421 (1991)).
  • 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
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the 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 (McCafferty, 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 a polypeptide sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO
  • 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).
  • n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • the 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. et al. (1993) Anticancer Drug Des. 8:53-63).
  • a nucleic acid molecule which encodes a polypeptide of this invention may be identical to the coding sequence of one or more of the nucleic acid
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO.14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:
  • nucleic acid molecules 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.
  • nucleic acid molecules of the second and third aspects of the invention may also encode the functional equivalents of the polypeptides of the first aspect of the invention.
  • a nucleic acid molecule may be a naturally-occurring variant such as a naturally- occurring allelic variant, or the molecule may be a variant that is not known to occur naturally.
  • non-naturally occurring variants of the 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.
  • 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
  • antisense molecules such as oligonucleotides
  • oligonucleotides can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the 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 (15OmM NaCl, 15mM trisodium citrate), 5OmM 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 0.1 X 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.
  • nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INSP 191 polypeptides and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • 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 such coding sequences, or is a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98%, 99% or more 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 INSPl 91 polypeptides.
  • 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 INSP 191 polypeptides 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 INSP 191 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).
  • 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:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,
  • 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.
  • telomere shortening uses universal primers to retrieve unknown nucleic acid sequence adjacent a known locus.
  • Inverse PCR may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
  • capture PCR 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
  • 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 of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the 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 ah, ⁇ 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 of the 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 ah, Basic Methods in Molecular Biology (1986) and Sambrook et ah, (supra). Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et ah, 1989 [supra]; Ausubel et ah, 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 of the 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 of the translated polypeptide into the lumen of the 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 of the expression of the polypeptide relative to the growth of the 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 (Stratagene, 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. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • the control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • 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
  • CHO Chinese hamster ovary
  • BHK baby hamster kidney
  • COS monkey kidney
  • C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac" kit).
  • host cells 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 known 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 1 1 :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, F. et al. (1981) J. MoI. 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.
  • transformed cells containing the appropriate sequences can be identified by the absence of marker gene function.
  • 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 of the 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.
  • DNA- DNA or DNA-RNA hybridizations 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 radionucleides, 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 of the polypeptides of the 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.
  • 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 immunoaff ⁇ nity techniques.
  • FACS fluorescence activated cell sorting
  • the medium can be recovered in order to recover and purify the expressed polypeptide.
  • polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • the present invention also provides novel targets and methods for the screening of drug candidates or leads. These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals.
  • a particular object of this invention resides in the use of an INSP191 polypeptide as a target for screening candidate drugs for treating or preventing complement related disorders.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a INSPl 91 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a INSP 191 polypeptide with a candidate compound, and selecting compounds that bind said INSP 191 polypeptide at the surface of said cells and/or that modulate the activity of the INSP 191 polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound having complement activity, and further preferably a compound that can be used for treating INSP 191 related disorders, or as a lead to develop drugs for treating complement related disorder.
  • a “biologically active” compound preferably is a compound that modulates the activity of INSP191.
  • the above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of complement related disorder, such as an animal model.
  • Preferred selected compounds are agonists of INSP 191, i.e., compounds that can bind to INSPl 91 and mimic the activity of an endogenous ligand thereof.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSPl 91 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INSP 191 polypeptide.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 191 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INSP 191 gene, preferably to stimulate expression thereof.
  • this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a INSPl 91 gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.
  • the polypeptide of the 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 of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the 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).
  • Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to complement related disorder in a subject.
  • the determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc.
  • the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
  • Modulation of activity includes, without limitation, stimulation of the surface expression of the INSPl 91 receptor, modulation of multimerization of said receptor ⁇ e.g., the formation of multimeric complexes with other sub-units), etc.
  • the cells used in the assays may be any recombinant cell ⁇ i.e., any cell comprising a recombinant nucleic acid encoding a INSPl 91 polypeptide) or any cell that expresses an endogenous INSP191 polypeptide. Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • E.coli E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines ⁇ e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures ⁇ e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
  • mammalian cell lines ⁇ e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • 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 of the 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 of the 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 of the present invention comprises:
  • a particular example is cotransfecting a construct expressing a polypeptide according to the invention, or a fragment such as the LBD, in fusion with the GAL4 DNA binding domain, into a cell together with a reporter plasmid, an example of which is pFR-Luc (Stratagene Europe, Amsterdam, The Netherlands).
  • This particular plasmid contains a synthetic promoter with five tandem repeats of GAL4 binding sites that control the expression of the luciferase gene. When a potential ligand is added to the cells, it will bind the GAL4-polypeptide fusion and induce transcription of the luciferase gene.
  • the level of the luciferase expression can be monitored by its activity using a luminescence reader (see, for example, Lehman et al. JBC 270, 12953, 1995; Pawar et al. JBC, 277, 39243, 2002).
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises: (a) contacting a labelled or unlabeled compound with the polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself; or
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the 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 of the present invention comprises: determining the inhibition of binding of a ligand to cells which express a polypeptide of the invention (and which optionally have a polypeptide of the 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.
  • 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.
  • step (c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, 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 immobilized polypeptide or the whole cell or the cell membrane after step (c); and
  • the polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose-dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
  • 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 of the manipulated cells. For example, such experiments reveal details of signaling 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).
  • 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 and spectroscopy.
  • 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.
  • this invention relates to the use of a INSP 191 polypeptide or fragment thereof, whereby the fragment is preferably a INSPl 91 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP 191 polypeptide for the treatment of an immune related disorder, wherein said agonist or stimulator is selected from the group consisting of:
  • a specific antibody or fragment thereof including: a) a chimeric, b) a humanized or c) a fully human antibody, as well as;
  • an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • a fibronectin-based binding molecule e.g. trinectin or adnectin.
  • Anticalins are also known in the art (Vogt et al., 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224.
  • test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
  • an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. tri
  • 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 of the invention discovered by the methods that are described above.
  • the various moieties of the invention ⁇ i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention) may be useful in the therapy or diagnosis of diseases.
  • 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.
  • compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted 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.
  • 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 of the 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 B.I. 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. Alternatively the 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 of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the 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 of the 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 contrast, in vivo gene therapy does not require isolation and purification of a patient's cells.
  • 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
  • AAV adeno-associated virus
  • a nucleic acid molecule encoding a polypeptide of the 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.
  • 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
  • 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 ⁇ i.e. to prevent infection) or therapeutic (i.e. 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, H. 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 antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the 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 of the 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.
  • This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the 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 al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al. , J. Virol. Meth., 35, 1 17-126 (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 of the 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 of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the 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 of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the 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 of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the 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 al., Genomics, 5, 874-879
  • 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 radiolabeled 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. Further, 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 al., Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
  • mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al., DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane.
  • Fluorescence in situ hybridization is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al., Science, 250, 559-562 (1990), and Trask et al., Trends, Genet., 7, 149-154 (1991)).
  • 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 al., Science (1996), VoI 274, pp 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al.); 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 W095/251 16 (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 of the 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 of the 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.
  • reporter molecules A wide variety of reporter molecules known in the art may be used, several of which are described above. Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • 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 of the present invention may comprise: (a) a nucleic acid molecule of the present invention
  • 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 disease in which complement proteins are implicated.
  • the disease is selected from infectious disease, glomerular disease, cardiovascular disease or hematology-related disorder or other disorder.
  • infectious disease is selected from Systemic Fungal Disease, Rickettsial Disease, Chlamydial Disease, Parasitic Infection, Viral Disease, Abscess, Human Immunodeficiency Virus Infection, Bacteremia, Septic Shock, sexually Transmitted Disease or Bacterial Disease.
  • the bacterial disease is selected from a disease caused by Gram-Positive Cocci, caused by Gram-Negative Aerobic Cocci, caused by Gram-Positive Bacilli, caused by Gram-Negative Bacilli, caused by Anaerobic Bacilli, caused by Spirochetes or caused by Mycobacteria.
  • the disease caused by Gram-Negative Aerobic Cocci is selected from meningitis, bacteremia, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrheal infection, acute cacterial meningitis or Meningococcal infection.
  • the Parasitic Infections is selected from Extraintestinal Protozoa infection, infection with Free-Living Amebas, Intestinal Protozoa infection, Nematode (Roundworm) Infection, Trematode (Fluke) infection or Cestodes (Tapeworms) infection.
  • the Extraintestinal Protozoa infection is malaria.
  • the viral disease is selected from Respiratory Viral Disease, Herpesvirus Infection, Central Nervous System Viral Disease, Arbovirus or Arenavirus Disease.
  • the glomerular disease is selected from nephritic syndrome, nephrotic syndrome, primary glomerular disease, or secondary renal disease.
  • the primary glomerular disease is selected from minimal change disease, focal segmental glomerulosclerosis, membranous glomerulonelhritis, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, IgA nephropathy, rapidly progressive glomerulonephritis, or fibrillary glomerulonephritis.
  • the nephritic syndrome is selected from hematuria, hypertension, renal insufficiency, edema, acute glomerulonephritis, transient glomerulonephritis, postinfectious glomerulonephritis, fulminant glomerulonephritis, rapidly progressive glomerulonephritis (RPGN), indolent Glomerulonephritis, IgA nephropathy, Crescentic glomerulonephritis, Pauci-immune RPGN, Immune complex RPGN, Anti-GBM antibody disease autoimmunity, primary renal hematuric-proteinuric syndrome, asymptomatic hematuric-proteinuric syndrome, chronic nephritic-proteinuric syndrome, chronic glomerulonephritis, slowly progressive glomerular disease.
  • RPGN rapidly progressive glomerulonephritis
  • the cardiovascular disorder is selected from Cardiac and Respiratory Arrest, Valvular Heart Disease, Arterial Hypertension, Endocarditis, Orthostatic Hypotension, Syncope, Pericardial Disease, Arteriosclerosis, Cardiac Tumor, Coronary Artery Disease, Disease of the Aorta and Its Branches, Heart Failure, Peripheral Vascular Disorder, Shock, Athletic Heart Syndrome or Arrhythmia.
  • the hematology-related disorder is selected from Anemia, Histiocytic Syndrome, Iron Overload related disorder, Leukemia, Lymphoma, Myeloproliferative Disorder, Plasma Cell Dyscrasia, Hemostasis and Coagulation Disorder, Disorder of the Spleen, Thrombotic Disorder, Platelet Disorder, Vascular Bleeding Disorder, Leukopenia, Lymphocytopenia or AIDS- Associated Hematologic Disorder and Malignancy.
  • the other disorder is selected from Alzheimers, multiple sclerosis, systemic lupus erythematosus or liver cancer, especially hepatocellular carcinoma.
  • Figure 1 Top 10 BLASTP hits for INSP 191 polypeptide sequence (SEQ ID NO:84) against NCB I- nr
  • FIG. 4 Signal peptide prediction (SignalP V2.0) for INSPl 91 polypeptide sequence (SEQ ID NO: 84)
  • Example 1 INSP 191 Protein BLAST Results
  • the INSPl 91 polypeptide sequence (SEQ ID NO: 84) was used as a protein BLAST query sequence against the NCBI non-redundant sequence database.
  • Figure 1 shows the top ten c ⁇ >
  • Example 2 CDD output Figure 3 shows the CDD output for the INSP 191 polypeptide sequence (SEQ ID NO: 84). The sequence is shown as containing alpha-2-macroglobulin, netrin and anaphylatoxin domains. This is characteristic of the complement proteins C3, C4 and C5.
  • Example 3 INSP191 signal sequence
  • Figure 4 shows that INSP191 is predicted to possess a signal peptide at the start of the protein.
  • the signal peptide cleavage site is thought to be between residues 21 and 22 of the INSPl 91 polypeptide sequence (Nielsen, H. et al. 1997, Protein Engineering, 10, 1-6; Nielsen, H., and Krogh, A.: Prediction of signal peptides and signal anchors by a hidden Markov model. In Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California, pp. 122-130 (1998)).
  • Human cDNA templates for example, from a variety of normal human tissue total RNA samples (which can either be purchased from companies such as Clontech, Stratagene, Ambion, Biochain Institute or prepared in-house) using an enzyme such as Superscript II RNase H- Reverse Transcriptase (Invitrogen).
  • Human cDNA libraries in bacteriophage lambda ( ⁇ ) vectors can be purchased from Clontech, Invitrogen, or made in-house in ⁇ GTlO vectors.
  • Pairs of specific PCR primers can be designed for amplifying the complete coding sequence of the virtual cDNA using software such as the 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 OC and a GC content of 40-60%. Primers should be selected which have high selectivity for the target sequence (INSP191) with little or no non-specific priming. PCR can then be used to amplify the gene sequence of interest.
  • the presence of the transcripts for INSP 191 may be investigated by PCR of cDNA from different human tissues.
  • the INSP 191 transcripts may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result.
  • all RNA should be treated with DNAse prior to use for reverse transcription.
  • a control reaction may be set up in which reverse transcription was not undertaken (a -ve RT control). For example, 1 ⁇ g of total 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 (-ve RT control).
  • PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls.
  • INSP 191 -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 INSPl 91 transcripts, not only those generated as described above. The tissue distribution pattern of the INSP191 polypeptides will provide further useful information in relation to the function of those polypeptides.
  • overexpression or knock-down of the expression of the polypeptides in cell lines may be used to determine the effect on transcriptional activation of the host cell genome.
  • Dimerisation partners, co-activators and co-repressors of the INSP191 polypeptide may be identified by immunoprecipitation combined with Western blotting and immunoprecipitation combined with mass spectroscopy.
  • Example 5 Fulminant hepatitis assay
  • the proinflammatory mediators C3a and C5a are essential for liver regeneration (see, for example, Strey CW, Markiewski M, Mastellos D, Vietnamesean R, Spruce LA, Greenbaum LE, Lambris JD. J Exp Med. 2003 Sep 15;198(6):913-23).
  • the INSP191 protein may have a useful function in counteracting the effects of toxic liver disease.
  • ConA-induced liver toxicity is one of three experimental models of T-cell dependent apoptotic and necrotic liver injury described in mice.
  • Gal N D-Galactosamine sensitized mice challenged with either activating anti- CD3 monoclonal AB or with superantigen SEB develop severe apoptotic and secondary necrotic liver injury (Kusters S, Gastroenterology. 1996 Aug;l l 1(2):462-71). Injection of the T-cell mitogenic plant lectin ConA to non-sensitized mice results also in hepatic apoptosis that preceeds necrosis. ConA induces the release of systemic TNF ⁇ and IFN ⁇ and various other cytokines. Both TNF ⁇ and IFN ⁇ are critical mediators of liver injury. Transaminase release 8 hours after the insult indicates severe liver destruction.
  • mice were maintained in standard conditions under a 12-hour light-dark cycle, provided irradiated food and water ad libitum.
  • His or StrepII tagged hIL-6 or INSP 191 genes may be cloned in the Gateway compatible pDEST12.2 containing the CMV promoter.
  • mice are anaesthetised with gas (isofluran, Baxter, Ref: ZDG9623). Hindlimbs are shaved and an echo graphic gel applied. Hyaluronidase is injected in the posterior tibialis mucle with (2OU in 50 ⁇ l sterile NaCl 0.9%, Sigma, Ref. H3631). After 10 min, 100 ⁇ g of plasmid (50 ⁇ g per leg in 25 ⁇ l of sterile NaCl 0.9%) is injected in the same muscle. The
  • DNA was prepared in the Buffer PBS-L-Glutamate (6 mg/ml; L-Glutamate, Sigma, P4761) before intra-muscular injection.
  • the electric field is applied for each leg with the ElectroSquarePorator (BTX, ref ECM830) at 75 Volts during 20 ms for each pulse, 10 pulses with an interval of 1 second in a unipolar way with 2 round electrodes (size 0.5 mm diameter) (Mir LM et al, Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4262-7 and Haas K et al, Neuron. 2001 Mar;29(3):583-9L).
  • BTX ElectroSquarePorator
  • 100 ⁇ l of blood is sampled from the eye at various 1.30h, 6h and 8h time-points. At the time of sacrifice, blood is taken from the heart.
  • IL-2, IL-5, IL-4, TNF ⁇ and IFN ⁇ cytokine levels are measured using the TH1/TH2 CBA assay (BD 551287).
  • Aspartate AminoTransferase (ASAT), ALanine Amino Transferase ALAT and urea blood parameters are determined using the COBAS instrument (Hitachi).
  • CHO cell produced hIL-6 is injected 1 hour before ConA injection.
  • INSP 191 and IL-6 electrotransfer At day 0 electrotransfer of INSP 191 or hIL-6 vectors as well as the empty vector (negative control) is performed (according to the above protocol). At day 5 after electrotransfer, ConA (20mg/kg) is injected iv and blood sampled at 3 time-points (1.30, 6, 24 hours). Cytokines, ASAT and ALAT measurements are performed as described above. Results
  • Example 6 ESTs ESTs matching the genomic region of INSP 191 have been found to be located in the liver, in particular in hepatocellular carcinoma (see Table 3). INSP 191 has also been found to be expressed in muscle, CD4 + T cells and hematopoietic cells.

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Abstract

Cette invention concerne une nouvelle protéine, appelée INSP191, identifiée ici comme protéine sécrétée, en particulier, comme protéine du complément; l'invention concerne aussi l'utilisation de cette protéine et de la séquence d'acide nucléique provenant du gène codant dans le diagnostic, la prévention et le traitement d'une maladie.
PCT/GB2007/002334 2006-07-06 2007-06-25 Protéine du complément WO2008003925A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0613471.2 2006-07-06
GB0613471A GB0613471D0 (en) 2006-07-06 2006-07-06 Protein

Publications (2)

Publication Number Publication Date
WO2008003925A2 true WO2008003925A2 (fr) 2008-01-10
WO2008003925A3 WO2008003925A3 (fr) 2008-02-28

Family

ID=36926595

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/002334 WO2008003925A2 (fr) 2006-07-06 2007-06-25 Protéine du complément

Country Status (2)

Country Link
GB (1) GB0613471D0 (fr)
WO (1) WO2008003925A2 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001081578A2 (fr) * 2000-04-26 2001-11-01 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres
WO2002102321A2 (fr) * 2001-06-18 2002-12-27 Curagen Corporation Proteines et acides nucleiques les codant
US20040029220A1 (en) * 2000-04-26 2004-02-12 Vernet Corine A.M. Novel proteins and nucleic acids encoding same
WO2005049806A2 (fr) * 2003-03-14 2005-06-02 Nuvelo, Inc. Nouveaux acides nucleiques et polypeptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001081578A2 (fr) * 2000-04-26 2001-11-01 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres
US20040029220A1 (en) * 2000-04-26 2004-02-12 Vernet Corine A.M. Novel proteins and nucleic acids encoding same
WO2002102321A2 (fr) * 2001-06-18 2002-12-27 Curagen Corporation Proteines et acides nucleiques les codant
WO2005049806A2 (fr) * 2003-03-14 2005-06-02 Nuvelo, Inc. Nouveaux acides nucleiques et polypeptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAHU A ET AL: "Structure and biology of complement protein C3, a connecting link between innate and acquired immunity" IMMUNOLOGICAL REVIEWS, MUNKSGAARD, vol. 180, April 2001 (2001-04), pages 35-48, XP002962792 ISSN: 0105-2896 *

Also Published As

Publication number Publication date
WO2008003925A3 (fr) 2008-02-28
GB0613471D0 (en) 2006-08-16

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