WO2008032053A1 - Variantes d'épissure fhr-4a - Google Patents

Variantes d'épissure fhr-4a Download PDF

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Publication number
WO2008032053A1
WO2008032053A1 PCT/GB2007/003443 GB2007003443W WO2008032053A1 WO 2008032053 A1 WO2008032053 A1 WO 2008032053A1 GB 2007003443 W GB2007003443 W GB 2007003443W WO 2008032053 A1 WO2008032053 A1 WO 2008032053A1
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polypeptide
seq
nucleic acid
acid molecule
disease
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PCT/GB2007/003443
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English (en)
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Kinsey Maundrell
Mark Ibberson
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Ares Trading S.A.
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Publication of WO2008032053A1 publication Critical patent/WO2008032053A1/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

Definitions

  • the present invention provides novel FHR-4A splicing variants (INSP216 and INSP216sv) containing an additional Sushi/CCP domain (called CCPO), which is homologous to Sushi/CCP domain 5 of human Complement Factor H, at the N- 5 terminus.
  • the present invention also relates to a novel FHR-4A homologue (called INPS216var) containing five Sushi/CCP domains.
  • bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable 0 of outpurting results in which a high degree of confidence can be placed.
  • 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.
  • Complement factor H is a member of a family of proteins involved in the regulation of complement activation (RCA). These secreted proteins share a common structural motif, the Short Consensus Repeat (SCR, also known as Sushi or CCP domain), which is structurally conserved among related genes and between phylogenetically divergent species.
  • SCR Short Consensus Repeat
  • fH is composed of 20 such Sushi/CCP domains and a variety of biological functions have been localised as specific to such domains.
  • the Sushi/CCP domain is characterized by cysteines and other polar or hydrophobic residues distributed over about 60 amino acids and it is usually encoded by a single exon, and processes such as gene conversion, duplication and exon shuffling have been implicated in the evolution and genomic radiation of Sushi/CCP domains-encoding genes (Estaller C et al., Eur J Immunol. 1991, 21 :799-802; Ripoche J et al., Biochem J. 1988, 249: 593-602; Zipfel PF et al., Biochem Soc Trans. 2002, 30: 971-8; Krushkal J et al., MoI Biol Evol.
  • FHL-I Factor H-like protein
  • FHRs Factor H-related proteins
  • FHR-4 Skerka C et al., J Biol Chem. 1997, 272:5627-34
  • FHR-4A Jozsi M et al., Eur J Hum Genet. 2004 Nov 24, electronic publication ahead of print; SEQ ID NO: 1 in WO 99/18900).
  • diseases include auto-immune diseases including psoriasis, adult/acute respiratory distress syndrome, bullous pemphigoid, rheumatoid arthritis, systemic lupus erythematosus, ischemia-reperfusion injury (Allegretti M et al., Curr Med Chem, 12: 217-36, 2005; Carroll MC, Nat Rev Immunol, 4: 825-31, 2004; Molina H, Rheum Dis Clin North Am, 30:1-18, 2004); renal and glomerural diseases (Quigg RJ, Curr Dir Autoimmun, 7: 165-80, 2004); neurodegenerative, cerebrovascular and neuroinflammatory diseases including cerebral ischemia and trauma, Alzheimer's disease, multiple sclerosis (van Beek J et al., Ann N Y Acad Sci, 992:56-71, 2003; McGeer PL and McGeer EG, Trends MoI Med, 8:519-23, 2002; Gasque P et
  • the present invention provides novel FHR-4A splicing variants (herein after referred to as INSP216 and INSP216sv) containing an additional Sushi/CCP domain (called CCPO), which is homologous to Sushi/CCP domain 5 of human Complement Factor H, at the N-terminus, and to a novel FHR-4A homologue (herein after referred to as INSP216var).
  • INSP216 and INSP216sv additional Sushi/CCP domain
  • CCPO Sushi/CCP domain 5 of human Complement Factor H
  • INSP216var novel FHR-4A homologue
  • the invention provides a polypeptide, which polypeptide:
  • (ii) is a fragment thereof which is a CCPO domain-containing polypeptide or has an antigenic determinant in common with one or more of the polypeptides of (i) or;
  • a polypeptide according to the invention is a CCPO domain-containing polypeptide.
  • CCPO domain-containing polypeptide refers to a molecule containing at least one CCPO domain.
  • the "CCPO domain-containing polypeptide” may be a molecule containing a CPPO domain-containing protein detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
  • CCPO domain-containing polypeptide may be a molecule matching the HMM build of the Pfam entry detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
  • Preferred polypeptides according to this aspect of the invention comprise the amino acid sequence as recited in SEQ ID NO:24, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO: 44, SEQ ID NO: 46 or SEQ ID NO: 48.
  • the polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "the INSP216 exon 1 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:4 is referred to hereafter as "the INSP216 exon 2 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:6 is referred to hereafter as "the INSP216 exon 3 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 8 is referred to hereafter as "the INSP216 exon 4 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 10 is referred to hereafter as "the INSP216 exon 5 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 12 is referred to hereafter as "the INSP216 exon 6 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 14 is referred to hereafter as "the INSP216 exon 7 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 16 is referred to hereafter as "the INSP216 exon 8 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as "the INSP216 exon 9 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:20 is referred to hereafter as "the INSP216 exon 10 polypeptide”.
  • polypeptide having the sequence recited in SEQ ID NO:22 is referred to hereafter as "the INSP216 exon 11 polypeptide".
  • polypeptide having the sequence recited in SEQ ID NO:24 is referred to hereafter as "the INSP216 full length polypeptide”.
  • SEQ ID NO:24 is produced by combining SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22.
  • the polypeptide having the sequence recited in SEQ ID NO:24 is referred to hereafter as "the INSP216 full length polypeptide".
  • the sequence recited in SEQ ID NO:34 is a splice variant of INSP216, which is 10 amino acids shorter than SEQ ID NO:24.
  • the polypeptide having the sequence recited in SEQ ID NO:30 is referred to hereafter as "the INSP216svl exon 5 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:32 is referred to hereafter as "the INSP216svl exon 6 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:34 is referred to hereafter as "the INSP216svl full length polypeptide”.
  • SEQ ID NO:34 is produced by combining SEQ ID NOs: 2 and 4, 6, 8, 30, 32, 16, 18, 20 and 22.
  • SEQ ID NO:50 is produced by combining SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20 and 22
  • the polypeptide having the sequence recited in SEQ ID NO:26 is referred to hereafter as "the INSP216 mature exon 1 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:28 is referred to hereafter as "the INSP216 mature full length polypeptide”.
  • SEQ ID NO:28 is produced by combining SEQ ID NOs: 26 and 4, 6, 8, 10 , 14, 16, 18, 20 and 22.
  • the polypeptide having the sequence recited in SEQ ID NO:28 is referred to hereafter as "the INSP216 mature full length polypeptide". This sequence is identical to the SEQ ID No:24 without the signal peptide.
  • polypeptide which polypeptide:
  • (ii) is a fragment thereof which is a FHR-4A-like polypeptide or has an antigenic determinant in common with one or more of the polypeptides of(i) or;
  • the polypeptide of the invention is an FHR-4A-like polypeptide.
  • FHR-4A-like polypeptide refers to a molecule containing at least FHR-4A- like domain.
  • the "FHR-4A-like polypeptide” may be a molecule containing a FHR-4A- like domain with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
  • FHR-4A-like polypeptide may be a molecule matching the HMM build of the Pfam entry detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
  • Preferred polypeptides according to this aspect of the invention comprise the amino acid sequence as recited in SEQ ID NO:52.
  • the sequence recited in SEQ ID NO:52 is a variant of INSP216, hereinafter referred to as INSP216var.
  • INSP216var contains alternative exons 1, 3 and 4 and is missing exons 2, 5, 6, 7 and 9 of INSP216.
  • FNSP216var has exons 8, 10 and 1 1 in common with INSP216.
  • SEQ ID NO:52 is produced by combining SEQ ID NOs: 75, 77, 79, 81, 83 and 85.
  • 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.
  • the polypeptide having the sequence recited in SEQ ID NO: 36 is referred to hereafter as "the HIS tag INSP216 full length polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 38 is referred to hereafter as "the HIS tag INSP216 mature polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 40 is referred to hereafter as "the HIS tag INSP216svl polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 54 is referred to hereafter as "the HIS tag INSP216sv2 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 56 is referred to hereafter as "the HIS tag INSP216var polypeptide”.
  • the CCPO domain of INSP216, INSP216svl and INSP216sv2 is homologous to CCP5 of human Complement Factor H ( Figure 2), somehow completing the tandem structure of FHR-4A with a tenth Sushi/CCP domain, so it is possible that alternative sequences INSP216-Nt and INSP216sv-Nt including only CCP0-CCP4 exist and are complementary to sequence disclosed as FHR-4 in the literature (Skerka C et al., J Biol Chem. 1997, 272:5627-34).
  • the polypeptide having the sequence recited in SEQ ID NO: 42 is referred to hereafter as "the INSP216-Nt full length polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 44 is referred to hereafter as "the INSP216-Nt mature polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 46 is referred to hereafter as "the INSP216svl Nt full length polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 48 is referred to hereafter as "the INSP216svl Nt mature polypeptide”.
  • the polypeptide therefore comprises or consists of the amino acid sequence as recited in: SEQ ID NO:4, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO: 46, or SEQ ID NO:48.
  • SEQ ID NO:4 amino acid sequence as recited in: SEQ ID NO:4, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO: 46, or SEQ ID NO:48.
  • lipoproteins preferably TG-Lp and/or VLDL, or
  • One preferred assay is the binding assay for the complement component C3b using surface plasmon resonance as described in Hellwage et al. (FEBS Lett. 1999 Dec 3;462(3):345-52.).
  • 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 INSP216 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.
  • “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 INSP216 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 identical or higher activity compared with INSP216 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%, 99%, 100% or more activity compared with INSP216 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • 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%.
  • functional equivalents can be fully functional or can lack function in one or more activities.
  • variations can affect the function, for example, of the activities of the polypeptide that reflect its possession of a CCPO domain or an FHR-4A-like domain.
  • 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 has the nucleic acid sequence as recited in any one of SEQ ID NO:1 (encoding the INSP216 exon 1 polypeptide), SEQ ID NO:3 (encoding the INSP216 exon 2 polypeptide), SEQ ID NO:5 (encoding the INSP216 exon 3 polypeptide), SEQ ID NO:7 (encoding the INSP216 exon 4 polypeptide), SEQ ID NO:9 (encoding the INSP216 exon 5 polypeptide), SEQ ID NO: 1 1 (encoding the INSP216 exon 6 polypeptide), SEQ ID NO: 13 (encoding the INSP216 exon 7 polypeptide), SEQ ID NO: 15 (encoding the INSP216 exon 8 polypeptide), SEQ ID NO: 17 (encoding the INSP216 exon 9 polypeptide), SEQ ID NO: 19 (encoding the INSP216 exon 10 polypeptide), SEQ ID NO:21 (encoding the INSP216 exon 1 1 polypeptide), SEQ ID NO:
  • the invention provides a purified nucleic acid molecule which hydridizes 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 0 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, and which preferably inhibits the ability of a polypeptide of the first aspect of the invention.
  • 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, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.
  • Such compounds may be identified using the assays and screening methods disclosed herein.
  • 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.
  • 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 INSP216 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 INSP216 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against CCPO domain-containing polypeptide related disorders or FHR-4A-like polypeptide related disorders.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of CCPO domain-containing polypeptide related disorders or FHR-4A-like polypeptide related disorders, comprising determining the ability of a compound to bind to an INSP216 gene or polypeptide, or a fragment thereof.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of CCPO domain-containing polypeptide related disorders or FHR-4A-like polypeptide related disorders, comprising testing for modulation of the activity of an INSP216 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 INSP216 proteins are implicated.
  • diseases include disease of the immune system (e.g. an autoimmune disease), renal and glomerural diseases, neurodegenerative, cerebrovascular and neuroinflammatory diseases, cardiovascular diseases and other pathalogical conditions.
  • the methods of the invention may be used in the manufacture of a medicament for the treatment of certain diseases including, but not limited to diseases involving unregulated complement activation (Bohana-Kashtan O et al., MoI Immunol, 41:583-97, 2004; Tsokos GC and Fleming SD, Curr Dir Autoimmun, 7:149-64, 2004; Schmidt BZ and Colten HR, Immunol Rev, 178:166-76, 2000).
  • diseases involving unregulated complement activation Bohana-Kashtan O et al., MoI Immunol, 41:583-97, 2004; Tsokos GC and Fleming SD, Curr Dir Autoimmun, 7:149-64, 2004; Schmidt BZ and Colten HR, Immunol Rev, 178:166-76, 2000.
  • autoimmune diseases including psoriasis, adult/acute respiratory distress syndrome, bullous pemphigoid, rheumatoid arthritis, systemic lupus erythematosus, ischemia-reperfusion injury (Allegretti M et al., Curr Med Chem, 12: 217-36, 2005; Carroll MC, Nat Rev Immunol, 4: 825-31, 2004; Molina H, Rheum Dis Clin North Am,30:l-18, 2004); renal and glomerural diseases (Quigg RJ, Curr Dir Autoimmun, 7:165-80, 2004); neurodegenerative, cerebrovascular and neuroinflammatory diseases including cerebral ischemia and trauma, Alzheimer's disease, multiple sclerosis (van Beek J et al., Ann N Y Acad Sci, 992:56-71, 2003; McGeer PL and McGeer EG, Trends MoI Med, 8:519-23, 2002; Gasque P et al., Mo
  • the assays set forth in the Examples may also be useful for the identification of therapeutically useful moieties.
  • the moieties of the present invention may have particular utility in the therapy or diagnosis of disorders/diseases (the two terms are used interchangeably herein) of the immune system (e.g. an autoimmune disease), renal and glomerural diseases, neurodegenerative, cerebrovascular and neuroinflammatory diseases, cardiovascular diseases and other pathalogical conditions.
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over a period of time towards a control level is indicative of regression of disease.
  • One possible 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 control protein.
  • the invention provides 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 including diseases involving unregulated complement activation for example, auto-immune diseases including psoriasis, adult/acute respiratory distress syndrome, bullous pemphigoid, rheumatoid arthritis, systemic lupus erythematosus, ischemia-reperfusion injury; renal and glomerural diseases; neurodegenerative, cerebrovascular and neuroinflammatory diseases including cerebral ischemia and trauma, Alzheimer's disease, multiple sclerosis; cardiovascular diseases and other pathological conditions.
  • diseases involving unregulated complement activation for example, auto-immune diseases including psoriasis,
  • 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 INSP216 polypeptides are CCPO domain-containing proteins or or FHR-4A-like polypeptides and thus have roles in many disease states. Antagonists of the INSP216 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 using in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • 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 polypeptides 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).
  • the polypeptide of the invention comprising a sequence having at least 85% of homology with INSP216 is a fusion protein.
  • fusion proteins can be obtained by cloning a polynucleotide encoding a polypeptide comprising a sequence having at least 85% of homology INSP216 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 INSP216 polypeptide.
  • heterologous sequences that can be comprised in the soluble fusion proteins either at N- or at C-terminus, are the following: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc region), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences allowing purification by affinity chromatography.
  • Fc region immunoglobulin constant regions
  • heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in the fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, Appl Microbiol Biotechnol, 60: 523- 33, 2003).
  • 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 protein used in the examples can be purified by means of a hexa- histidine peptide fused at the C-terminus of INSP216.
  • 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 Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO.73) 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 (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 IgG 2 or IgG 4 , or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric 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.
  • 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 INSP216 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 INSP216 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.
  • Typical 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.
  • 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.
  • non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the CPPO domain- containing 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, 2002), or eliminated by modifying their sequence following known methods for selecting mutations for increasing protein stability, and correcting them (van den Burg B and Eijsink V, 2002; WO 02/05146, WO 00/34317, WO 98/52976).
  • 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-difiuoro-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).
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • polypeptides of the first aspect of the invention have a degree of sequence identity with the INSP216 polypeptides, or with active fragments thereof, of greater than 70% or 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98%, 98.5%, 99% or 99.5% 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 Biopendium search database may be used (see WO 01/67507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP216 exon polypeptides or the INSP216 full length polypeptide (SEQ ID NOs. 24 and 28), are predicted to contain SUSHI domains, by virtue of sharing significant structural homology with the INSP216 exon polypeptides or the INSP216 polypeptide or INSP216 mature polypeptide.
  • the Inpharmatica Genome ThreaderTM predicts two proteins, or protein regions, to share structural homology with a certainty of at least 10% more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and above.
  • the certainty value of the Inpharmatica Genome ThreaderTM is calculated as follows. A set of comparisons was initially performed using the Inpharmatica Genome ThreaderTM exclusively using sequences of known structure. Some of the comparisons were between proteins that were known to be related (on the basis of structure). A neural network was then trained on the basis that it needed to best distinguish between the known relationships and known not-relationships taken from the CATH structure classification (www.biochem.ucl.ac.uk/bsm/cath).
  • polypeptides of the first aspect of the invention also include fragments of the INSP216 polypeptides and fragments of the functional equivalents of the INSP216 polypeptides, provided that those fragments contain a SUSHI domain or have an antigenic determinant in common with the INSP216 polypeptide or the INSP216 mature polypeptide.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP216 polypeptides or one of its functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant. Fragments according to the invention may be 1-100 amino acids in length, preferably, 5-50, more preferably 7-20 amino acids.
  • Nucleic acids according to the invention are preferably 10-1000 nucleotides in length, preferably 50-800 nucleotides, preferably 100-600, preferably 200-550, preferably 300- 500 nucleotides in length.
  • Polypeptides according to the invention are preferably 5-500 amino acids in length, preferably 50-400, preferably 100-300, preferably 150-250 amino acids in length.
  • Fragments of the full length INSP216 polypeptides may consist of combinations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 neighbouring exon sequences in the INSP216 polypeptide sequences, respectively. These exons may be combined with further mature fragments according to the invention. For example, such combinations include exons 1 and 2, and so on. Such fragments are included in the present invention. Fragments may also consist of combinations of different domains of the INSP216 protein. For example a fragment may consist of combinations of the different extracellular domains of INSP216 as recited above.
  • fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the fragment of the invention When comprised within a larger polypeptide, 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 other related polypeptides in the prior art such as known SUSHI domain-containing 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 other related polypeptides in the prior art.
  • polypeptide of the invention there is a measurable increase in the affinity for a polypeptide of the invention as compared with known CCPO domain-containing proteins or FHR-4A-like polypeptides.
  • polypeptide of the invention there is a measurable increase in the affinity for a polypeptide of the invention as compared with natural CCPO domain-containing proteins or FHR-4A-like polypeptides.
  • 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, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
  • 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)).
  • 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
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode the polypeptide sequences recited in SEQ ID NO:2, 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: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, 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).
  • 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 the polypeptide of SEQ ID NO: 2 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 1.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:4 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:3.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 6 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:5.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:8 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:7.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 10 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:9.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 12 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:11.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 14 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 13.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 16 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 15.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 18 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 17.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:20 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 19.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:22 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:21.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:24 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:23.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:26 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:25.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:28 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:27.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:30 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:29.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:32 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:31.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:34 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:33.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:36 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:35.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:38 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:37.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:40 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:39.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:42 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:41.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:44 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:43.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:46 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:45.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:48 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:47.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:50 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:49.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:52 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:51.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:54 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:53.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:56 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:55.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:75 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:74.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:77 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:76.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:79 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:78.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:81 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:80.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 83 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 82.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 85 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:84.
  • 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: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, 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
  • nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect 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 svs, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
  • antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide 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 0.1X SSC at approximately 65°C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding an INSP216 polypeptide (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: 75, SEQ ID NO: 77, SEQ ID
  • 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 a nucleic acid molecule having the sequence given in 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, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98%, 98.5%, 99% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSP216 full length polypeptide or the INSP216 full length mature polypeptide ot INSP216 var polypeptide.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INSP216 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding these polypeptides.
  • 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 INSP216 polypeptides is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989, 1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1 1, 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, SEQ ID NO: 74, SEQ ID NO: 76
  • Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation.
  • a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation.
  • Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.
  • RNA interference (Elbashir, SM et al, Nature 2001, 411, 494-498) is one method of sequence specific post-transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.
  • the vectors 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 al., (supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • HACs Human artificial chromosomes
  • PACJNSP216EC-6HIS and pDEST12.2_INSP216EC-6HIS are preferred examples of suitable vectors for use in accordance with the aspects of this invention relating to INSP216.
  • 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 al., Basic Methods in Molecular Biology (1986) and
  • 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, LaJoIIa, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • 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
  • baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells. There are many plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30, 3861-3863 (1991).
  • 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.
  • 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. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators 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 INSP216 polypeptide as a target for screening candidate drugs for treating or preventing CCPO domain-containing protein related disorders or FHR-4A-like polypeptide related disorders.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a INSP216 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 INSP216 polypeptide with a candidate compound, and selecting compounds that bind said INSP216 polypeptide at the surface of said cells and/or that modulate the activity of the INSP216 polypeptide.
  • a "biqlogically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound having CCPO domain activity, and further preferably a compound that can be used for treating INSP216 related disorders, or as a lead to develop drugs for treating CCPO domain- containing protein related disorders or FHR-4A-like polypeptide related disorders.
  • a "biologically active” compound preferably is a compound that modulates the activity of INSP216 or INSP216var.
  • 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 CPPO domain-containing protein related disorders or FHR-4A-like polypeptide related disorders, such as an animal model.
  • Preferred selected compounds are agonists of INSP216 or INSP216var, i.e., compounds that can bind to INSP216 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 INSP216 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INSP216 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 INSP216 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INSP216 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 INSP216 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 CCPO domain containing protein related disorders or FHR-4A-like polypeptide related disorders 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 INSP216 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 INSP216 polypeptide) or any cell that expresses an endogenous INSP216 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.
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • a method such as FRET detection of ligand bound to the polypeptide in the presence of peptide co-activators might be used.
  • 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:
  • 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;
  • step (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c);
  • 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;
  • step (d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c);
  • 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.
  • 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.
  • 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 signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance 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 INSP216 polypeptide or fragment thereof whereby the fragment is preferably a INSP216 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP216 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).
  • Anticalins are also known in the art (Vogt et al., 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224. Furthermore, the 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,
  • 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
  • the various moieties of the invention may be useful in the therapy or diagnosis of diseases.
  • one or more of the following assays may be carried out.
  • test compound refers to the test compound as being a protein/polypeptide
  • test compound a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound”.
  • compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier 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%. 98.5% or even 99% by weight.
  • the pharmaceutical 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 targetted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier 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
  • 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.
  • 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, intraperitoneal ⁇ , 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.
  • 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.
  • inhibition can be achieved using "triple helix" base-pairing methodology. 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.
  • the complementary sequence or antisense molecule may also be designed to block translation of niRNA 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. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
  • 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 vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479.
  • a nucleic acid molecule encoding a polypeptide 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.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches,- (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, 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 anti-oxidants, 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. For example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation. Genetic delivery of antibodies that bind to polypeptides according to the invention may also be effected, for example, as described in International patent application WO98/55607.
  • jet injection see, for example, www.powderject.com
  • jet injection may also be useful in the formulation of vaccine compositions.
  • This invention also relates to the use of nucleic acid molecules according to 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, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of:
  • a further aspect of the invention comprises a diagnostic method comprising the steps of:
  • 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 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with 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.
  • 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 ah, 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.
  • 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.
  • a wide variety of reporter molecules known in the art may be used, several of which are described above.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise:
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease, particularly certain diseases including, but not limited to, diseases involving unregulated complement activation for example, auto-immune diseases including psoriasis, adult/acute respiratory distress syndrome, bullous pemphigoid, rheumatoid arthritis, systemic lupus erythematosus, ischemia-reperfusion injury; renal and glomerural diseases; neurodegenerative, cerebrovascular and neuroinflamrnatory diseases including cerebral ischemia and trauma, Alzheimer's disease, multiple sclerosis; cardiovascular diseases and other pathological conditions.
  • diseases involving unregulated complement activation for example, auto-immune diseases including psoriasis, adult/acute respiratory distress syndrome, bullous pemphigoid, rheumatoid arthritis, systemic lupus erythematosus, ischemia-reperfusion injury; renal and glomerural diseases; neurodegenerative, cerebrovascular and neuroinflamrnatory diseases including cerebral
  • Figure 1 Multiple alignment of the protein sequence for human Complement Factor Pi- Related protein 4 (FHR4 HUMAN; SWISSPROT Ace. No. Q92496), human Complement Factor H-Related protein 4A (FHR-4A; SWISSPROT Ace. No. AJ640130; DERWENT DGENE Ace. No. AAY09065), INSP216 (SEQ ID NO: 24), and INSP216sv (SEQ ID NO: 34) with the indication of identical residues (*), of the predicted signal sequence (residues 1-18; boxed), of the predicted Sushi / CCP domain (numbered, within arrows), and of the predicted N-glycosylation sites (underlined).
  • Sushi/CCP domain common to INSP216, INSP216sv, and FHR-4A (rCCPl-rCCP9) with specific Sushi/CCP domains in human Complement Factor H (fCCP6, fCCP8, fCCP9, fCCP19, and fCCP20).
  • fCCP6, fCCP8, fCCP9, fCCP19, and fCCP20 Identical residues are indicated with * (the cysteines are also highlighted). The conserved residues are indicated with +.
  • Figure 3 alignment of INSP216 protein sequence with the human genomic DNA encoding this protein (as determined on the basis of the assembly hgl7 of May 2004).
  • Figure 4 A. Coding region of INSP216sv cDNA. The position and orientation of primers used for RT (AS475), PCR amplification of the full length coding sequence (AS476-AS477) and internal sequencing of the cloned cDNA (AS562) are indicated by arrows. Start and stop codons are indicated in bold type. Underlined nucleotides denote 6 SNPs which differ from the prediction. They are (based on numbering of the original coding sequence): G19T (Val>Phe), G555T (Glu>Asp), A1220G (Lys>Arg), T1296C (silent), T1782C (silent), A1834G (Thr>Ala).
  • Figure 5 A. Coding region of INSP216var cDNA. The position and orientation of primers used for RT (AS475), PCR amplification of the full length coding sequence (AS476-AS477) are indicated by arrows. Start and stop codons are indicated in bold type.
  • FHR-4A has been characterised as containing a tandem arrangement of four Sushi/CCP domains, with an additional Sushi/CCP domain at the C-terminus, for a total of nine Sushi/CCP domains.
  • the signal peptide and each of these domains is encoded by a distinct exon, with intronic sequences that may be considered similar to CCP5 and CCP7 of human Complement Factor H (Jozsi M et al., Eur J Hum Genet. 2004 Nov 24, electronic publication ahead of print).
  • a specific assembly of genomic sequence generates a first open reading frame of 1914 nucleotides (SEQ ID NO: 23) encodes for a protein sequence called INSP216 comprising a protein or 648 amino acids (SEQ ID NO: 24).
  • This protein contains a predicted signal peptide (residues 1-18) leading a mature sequence, encoded by a DNA sequence of 1890 nucleotides (SEQ ID NO: 27), containing 630 amino acids (SEQ ID NO: 28), including 10 Sushi/CCP domains, with a first domain (CCPO; SEQ ID: 3 and 4) in addition to the nine characterizing human FHR-4A (CCP1-CCP9).
  • a differential splicing event may lead to the addition of 10 amino acids between CCP3 and CCP4, generating the sequence called INSP216sv (SEQ ID NO: 33 and 34).
  • INSP216 and INSP216sv are FHR-4 variants both characterized by the presence of CCPO, but sharing with this latter sequence other features, such as the signal peptide and the glycosylation sites (unless the one modified by the alternative splicing event in INSP216sv; Figure 1).
  • CCPO of INSP216 and INSP216sv is homologous to CCP5 of human Complement Factor H ( Figure 2), somehow completing the tandem structure of FHR-4A with a tenth Sushi/CCP domain, so that it can be suggested the possibility of alternative sequences INSP216-Nt (SEQ ID NO: 41-44) and INSP216sv-Nt (SEQ ID NO: 45-48) including only CCP0-CCP4 and complementary to sequence disclosed as FHR-4 in the literature (Skerka C et al., J Biol Chem. 1997, 272:5627-34). In fact, CCPO results from the inclusion of an additional exon, for a totality of 1 1 exons (Figure 3).
  • Table 3 summarizes the different features of the splice variants.
  • the table includes the domain boundaries of the signal peptide and the CCP domains, and the location of the glycosylation sites and the conserved cysteine residues.
  • transcripts including exons 2-6 can be identified and/or cloned by primer hybridization or by PCR.
  • INSP216 encodes a protein of 648 amino acids spanning 11 coding exons. It is a member of the Factor H-related proteins and is predicted to contain 9 sushi domains.
  • the strategy used to clone INSP216 cDNA was to prepare an initial pool of RNAs from a wide variety of human tissues (section 2.1.1) and from this to make a single preparation of multi-tissue polyA+ mRNA as template for reverse transcription.
  • Gene specific cDNA primers were designed for a small set of protein predictions (typically 5- 10 sequences), and aliquots of the resulting cDNA mix provided templates for separate PCR reactions using primers designed to obtain the corresponding coding region. Amplified fragments were then purified by gel electrophoresis and cloned into the Bluescript cloning vector by virtue of specific restriction sites added to the ends of the PCR primers.
  • the 1.8Kb fragment has an in-frame deletion of exon 2 which encodes the 1st sushi domain, and a second 30bp deletion around the junction between exons 5 and 6 which results from the use of alternative splice donor and acceptor sites. This latter deletion removes 10 amino acids and falls between (but leaves intact) sushi domains 3 and 4.
  • the 1 kb fragment encodes a 5 sushi domain protein which is encoded approximately 120 kb upstream in the chromosome (see Figure 5). This clone is therefore not considered to be a splice variant of the prediction but rather another member of the family and is therefore designated INSP216 variant.
  • the coding region and position of the oligonucleotide primers used in the cloning and sequencing of INSP216sv are shown in Figure 3 A.
  • the coding region and position of the oligonucleotide primers used in the cloning of INSP216var are shown in Figure 4A.
  • a preparation of human RNA was prepared by mixing approximately 10 ⁇ g total RNA from each of the following sources:
  • RNA was fractionated by chromatography on a pre-packed oligo-dT column (Stratagene) according to the protocol supplied by the manufacturer. Approximately 400 ⁇ g total RNA yielded 16 ⁇ g polyA+ mRNA which was aliquotted and frozen at -80 0 C.
  • the gene specific cDNA primer for INSP216 (AS475) was pooled with gene specific cDNA primers for 7 other protein predictions, each at a final concentration of IpM.
  • the pooled cDNA primer set was diluted 10 fold into 40 ⁇ l of a mixutre containing 1 x RT buffer, ImM each dNTPs, lU/ ⁇ l Protector RNA Inhibitor (Roche) and 1 ⁇ g denatured polyA+ RNA prepared as described above.
  • cDNA synthesis was initiated by addition of 2OU Transcriptor reverse transcriptase (Roche) and allowed to proceed for Ih at 50 °C. At the end of the reaction, 5 ⁇ l of the cDNA mix was used for PCR amplification as described below.
  • Top strand (AS476) and bottom strand (AS477) PCR primers were designed to span the entire predicted coding sequence of INSP216. BamHI restriction sites were added at the 5' end of each primer since no internal sites for this enzyme were predicted.
  • a reaction mixture (lOO ⁇ l) was set up containing 1 x PCR buffer 1, 0.35mM each dNTP, 0.3 ⁇ M each PCR primer, 5 ⁇ l cDNA above, and the PCR reaction was initiated by addition of 7.5U Expand long template enzyme (Roche).
  • Cycling conditions for 'touchdown' PCR were: 94 °C 2 min (I cycle); 94 °C 10 sec, 64 °C (decreasing by 1 °C each cycle) 30 sec, 68 °C 210 sec (14 cycles); 94 °C 10 sec, 50 °C 30 sec, 68 °C 210 + 5 sec/cycle (25 cycles); 68 0 C 7 min (1 cycle).
  • An aliquot of the PCR reaction was analysed by electrophoresis in a 0.8% agarose gel and found to contain 2 major fragments of 1.8kb and 1.Okb. The remainder of the PCR reaction was purified using the Wizard PCR Cleanup System (Promega) as recommended by the manufacturer, prior to subcloning of the PCR products.
  • Bluescript BSK- cloning vector was digested with BamHI and dephosphorylated using calf intestinal alkaline phosphatase (Roche Diagnostics) according to the supplier's recommendations.
  • the full length linearized and dephosphorylated cloning vector was separated on a 0.8% agarose gel, excised and purified using the Wizard Cleanup System (Promega) according to the protocol provided by the manufacturer.
  • the purified vector DNA and PCR products were mixed in a molar ratio of 1 :3 and precipitated overnight at -20 0 C in the presence of 2.5 volumes ethanol.
  • the precipitated DNA was recovered by centrifugation, washed in 70% ethanol, dried under vacuum and ligated in a final volume of 1 O ⁇ l using the Rapid Ligation Kit (Roche Diagnostics) according to the protocol supplied by the manufacturers.
  • the ligation mixture was then used to transform E. coli strain JMlOl as follows: 50 ⁇ l aliquots of competent JMlOl cells were thawed on ice and l ⁇ l or 5 ⁇ l of the ligation mixture was added. The cells were incubated for 40 min on ice and then heat shocked by incubation at 42 0 C for 2min. 1ml of warm (room temperature) L-Broth (LB) was added and samples were incubated for a further 1 h at 37 °C. The transformation mixture was then plated on LB plates containing ampicillin (100 ⁇ g/ml) IPTG (0.1 ⁇ M) and X-gal (50 ⁇ g/ml) and incubated overnight at 37 °C. Single white colonies were chosen for plasmid isolation.
  • Miniprep plasmid DNA was prepared from 5 ml cultures using a Biorobot 8000 robotic system (Qiagen) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices). Aliquots of miniprep plasmid DNAs (100-200ng) were digested with BamHI for 2h at 37 0 C and analysed by electrophoresis in 0.8% agarose gels. Plasmids with inserts of the expected sizes of either 1.8kb or 1.0kb were selected for DNA sequence analysis.
  • Inserts were sequenced from either end by mixing 200-500 ng plasmid DNA with either the T7 or T3 sequencing primers (see Table 4); full sequence of selected inserts was obtained in the same way using a custom-designed internal sequencing primer (AS 562 see Table 4). Sequencing reactions were processed using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Products were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) and analyzed on an Applied Biosystems 3700 sequencer.
  • the predicted mRNA coding sequence for INSP216 is 1944 nucleotides long and encodes a protein of 648 amino acids.
  • RT-PCR to obtain the coding sequence generated 2 fragments of approximately 1.8 Kb and 1.0 Kb both of which were cloned and sequenced.
  • the 1.8 kb fragment was found to encode an alternatively spliced variant of INSP216 which lacks exon 2 and which uses alternative splice donor and splice acceptor sites at the junction between exons 5 and 6. This results in a precise deletion of the first sushi domain encoded by exon 2, and deletion of a 10 amino acid sequence between sushi domains 3 and 4 which leaves the adjacent sushi domains intact.
  • Miniprep #14 was selected for further work.
  • SNPs were found of which 4 resulted in an altered polypeptide sequence compared the Inpharmatica prediction. They are (based on numbering of the original coding sequence): G19T (Val>Phe), G555T (Glu>Asp), A1220G (Lys>ArgR), T1296C (silent), T1782C (silent), A1834G (Thr>Ala).
  • the polypeptide sequence encoded by INSP216sv miniprep # 14 is shown in Figure 4B.
  • the ORF of INSP216SV was contained in the plasmid pBSK. Incorporation of Kozak sequence (GCC ACC), C-terminal 6HIS tag and stop codon were all accomplished by including the appropriate nucleotides in the primers used for PCR amplification.
  • Plasmid containing the INSP216SV sequence was used as PCR template to generate the full - length ORF containing a C-terminal 6HIS tag and a stop codon.
  • the first stage of this Gateway cloning process involved a two step PCR reaction which generates the full - length ORF of INSP216SV flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in- frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA).
  • 6HIS in- frame 6 histidine
  • the first PCR reaction PCRl (in a final volume of 50 ⁇ l) contains respectively: 1 ⁇ l (25 ng) of plasmid containing the INSP216SV sequence, 4.0 ⁇ l dNTPs (10 mM), 5 ⁇ l of 1OX Pfx polymerase buffer, 1.5 ⁇ l MgSO4 (50 mM), 1.0 ⁇ l each of gene specific primer (to give a final concentration of 100 pico-moles) (INSP216SV attB FP and INSP216SV attB RP), and 0.5 ⁇ l Platinum Pfx DNA polymerase (Invitrogen).
  • the PCR reaction was performed using an initial denaturing step of 94 °C for 2 min, followed by 30 cycles of 94 °C for 45 s; 60 0 C for 45 s and 68 0 C for 2 min; and a final extension cycle of 68 °C for 5 minutes and a holding cycle of 4 °C.
  • the second PCR reaction (in a final volume of 50 ⁇ l) contained 1 ⁇ l of diluted purified PCRl product (to a final concentration of 10 ng), 4.0 ⁇ l dNTPs (10 mM), 5 ⁇ l of 1OX
  • Pfx polymerase buffer 1.5 ⁇ l MgSO4 (50 mM), 1.0 ⁇ l of each Gateway conversion primer (to give a final concentration of 100 picomoles) (ATTB PCR FP [unique] and
  • the second stage of the Gateway cloning process involved sub cloning of the Gateway modified PCR product into the Gateway entry vector pDONR221 (Invitrogen) as follows: 5 ⁇ l of gel extracted product from PCR2 was incubated with 1.5 ⁇ l pDONR221 vector (0.1 ⁇ g/ ⁇ l), 2 ⁇ l BP buffer and 1.5 ⁇ l of BP clonase enzyme mix (Invitrogen) in a final volume of 10 ⁇ l at RT for Ih. The reaction was stopped by addition of 1 ⁇ l proteinase K (2 ⁇ g/ ⁇ l) and incubated at 37 0 C for a further 10 min.
  • DH5 ⁇ strain (Invitrogen) was used to transform DH5 ⁇ strain (Invitrogen) as follows: a 50 ⁇ l aliquot of DH5 ⁇ cells was thawed on ice and 2 ⁇ l of reaction mixture added. The mixture was incubated for 30 min on ice and then heat shocked by incubation at 42 °C for exactly 45 s. Samples were returned to ice and 250 ⁇ l of warm SOC media (room temperature) was added. Samples were incubated with shaking (250 rpm) for 1 h at 37 0 C. The transformation mixture was then plated on Luria Bertani (LB) plates containing kanamycin (50 ⁇ g/ml) and incubated overnight at 37 0 C.
  • LB Luria Bertani
  • the PCR mixture (in a final volume of 25 ⁇ l) contained 10 ⁇ l of the centrifuged cell lysate, 2.0 ⁇ l dNTPs (10 mM), 2.5 ⁇ l of Taq polymerase buffer, 0.5 ⁇ l of screening primers (to give a final concentration of 100 picomoles) (21 Ml 3 FP and ATTBl PCR RP [unique]) and 0.5 ⁇ l of Taq DNA polymerase.
  • the conditions for the screening PCR reaction were: 94 0 C for 2 min, followed by 30 cycles of 94 °C for 45 s; 58 °C for 45 s and 72 °C for 2 min; and a final extension cycle of 72 °C for 5 minutes and a holding cycle of 4 0 C.
  • the PCR products were loaded onto a 1.0 % agarose gel to verify the fragment size.
  • One positive clone was selected and plasmid mini-prep DNA was prepared from the 5 ml culture using QIAprep Spin Miniprep kit (Qiagen).
  • Plasmid DNA (150-200 ng) was subjected to DNA sequencing with 21M13 and M13Rev primers using the Applied Biosystems BigDye Terminator V 3.1 Cycle sequencing Kit (Applied Biosystems P/N 4336917) according to the manufacturer's instructions.
  • the primer sequences are shown in Table 4. Sequencing reactions were analyzed on ABI 3100 Genetic Analyzer (Applied Biosystems P/N 628-0030). After sequence confirmation of the insert, pDONR221_INSP216SV-6HIS, was then used to create the expression clones.
  • Plasmid DNA (2 ⁇ l or approx. 150 ng) of pDONR221JNSP216SV-6HIS was then used in a recombination reaction containing 1.5 ⁇ l of either pEAK12d vector or pDEST12.2 vector (0.1 ⁇ g / ⁇ l), 2 ⁇ l LR buffer and 1.5 ⁇ l of LR clonase (Invitrogen) in a final volume of 10 ⁇ l.
  • the reaction was stopped by addition of 1 ⁇ l proteinase K (2 ⁇ g/ ⁇ l) and incubated at 37 0 C for a further 10 min.
  • An aliquot of this reaction (2 ⁇ l) was used to transform DH5 ⁇ strain (Invitrogen) as follows: a 50 ⁇ l aliquot of DH5 ⁇ cells was thawed on ice and 2 ⁇ l of reaction mixture added. The mixture was incubated for 30 min on ice and then heat shocked by incubation at 42 0 C for exactly 45 s. Samples were returned to ice and 250 ⁇ l of warm SOC media (room temperature) was added. Samples were incubated with shaking (250 rpm) for 1 h at 37 °C. The transformation mixture was then plated on Luria Bertani (LB) plates containing Ampicillin (100 ⁇ g/ml) and incubated overnight at 37 °C.
  • LB Luria Bertani
  • the PCR mixture (in a final volume of 25 ⁇ l) contained 10 ⁇ l of the centrifuged cell lysate, 2.0 ⁇ l dNTPs (10 mM), 2.5 ⁇ l of Taq polymerase buffer, 0.5 ⁇ l of screening primers (to give a final concentration of 100 picomoles and 0.5 ⁇ l of Taq DNA polymerase.
  • pEAK12d clones were screened using the primers pEAK12 FP and pEAK12 RP and pDEST12.2 clones were screened using the primers 21M13FP and M13Rev RP.
  • the conditions for the screening PCR reaction were: 94 °C for 2 min, followed by 30 cycles of 94 °C for 45 s; 60 0 C for 45 s and 72 °C for 2 min; and a final extension cycle of 72 0 C for 5 minutes and a holding cycle of 4 °C.
  • the PCR products were loaded onto a 1.0 % agarose gel to verify the fragment size.
  • Plasmid mini-prep DNA was prepared from the 5 ml culture using QIAprep Spin Miniprep kit (Qiagen).
  • Plasmid DNA (150 - 200 ng) in the pEAK12d vector was subjected to DNA sequencing with the sequencing primers pEAK12 FP and pEAK12 RP as described above.
  • Plasmid DNA (150 - 200 ng) in the pDEST12.2 vector was subjected to DNA sequencing with the sequencing primers 2 IM 13 FP and M13Rev RP as described above.
  • the ORF of INSP216 variant (V) was contained in the plasmid pBSK. Incorporation of kozak sequence (GCC ACC), C-terminal 6HIS tag and stop codon were all accomplished by including the appropriate nucleotides in the primers used for PCR amplification.
  • Plasmid containing the ORF of the INSP216 variant (V) was used as PCR template to generate the full - length ORF containing a C-terminal 6HIS tag and a stop codon.
  • the first stage of this Gateway cloning process involved a two step PCR reaction which generates the full - length ORF of INSP216 variant flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in-frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA).
  • 6HIS in-frame 6 histidine
  • the first PCR reaction PCRl (in a final volume of 50 ⁇ l) contains respectively: 1 ⁇ l (25 ng) of plasmid containing the ORF of the INSP216 variant (V), 4.0 ⁇ l dNTPs (10 mM), 5 ⁇ l of 1OX Pfx polymerase buffer, 1.5 ⁇ l MgSO4 (50 mM), 1.0 ⁇ l each of gene specific primer (to give a final concentration of 100 pico- moles) (INSP216 [variant] attB FP and INSP216 [variant] attB RP), and 0.5 ⁇ l Platinum Pfx DNA polymerase (Invitrogen).
  • the PCR reaction was performed using an initial denaturing step of 94 0 C for 2 min, followed by 30 cycles of 94 °C for 30 s; 60°C for 30 s and 68 °C for 1 min; and a final extension cycle of 68 °C for 5 minutes and a holding cycle of 4 0 C.
  • the second PCR reaction (in a final volume of 50 ⁇ l) contained 1 ⁇ l of diluted purified PCRl product (to a final concentration of 10 ng), 4.0 ⁇ l dNTPs (10 mM), 5 ⁇ l of 1OX Pfx polymerase buffer, 1.5 ⁇ l MgSO4 (50 mM), 1.0 ⁇ l of each Gateway conversion primer (to give a final concentration of 100 picomoles) (ATTB PCR FP [unique] and ATTB PCR RP [unique]) and 0.5 ⁇ l of Platinum Pfx DNA polymerase.
  • the conditions for the 2nd PCR reaction were: 94 0 C for 2 min, followed by 30 cycles of 94 0 C for 30 s; 60 0 C for 30 s and 68 0 C for 1 min; and a final extension cycle of 68 °C for 5 minutes and a holding cycle of 4 0 C.
  • the second stage of the Gateway cloning process involved sub cloning of the Gateway modified PCR product into the Gateway entry vector pDONR221 (Invitrogen) as follows: 5 ⁇ l of gel extracted product from PCR2 was incubated with 1.5 ⁇ l pDONR221 vector (0.1 ⁇ g/ ⁇ l), 2 ⁇ l BP buffer and 1.5 ⁇ l of BP clonase enzyme mix (Invitrogen) in a final volume of 10 ⁇ l at RT for Ih. The reaction was stopped by addition of 1 ⁇ l proteinase K (2 ⁇ g/ ⁇ l) and incubated at 37 °C for a further 10 min.
  • DH5 ⁇ strain (Invitrogen) was used to transform DH5 ⁇ strain (Invitrogen) as follows: a 50 ⁇ l aliquot of DH5 ⁇ cells was thawed on ice and 2 ⁇ l of reaction mixture added. The mixture was incubated for 30 min on ice and then heat shocked by incubation at 42 0 C for exactly 45 s. Samples were returned to ice and 250 ⁇ l of warm SOC media (room temperature) was added. Samples were incubated with shaking (250 rpm) for 1 h at 37 0 C. The transformation mixture was then plated on Luria Bertani (LB) plates containing kanamycin (50 ⁇ g/ml) and incubated overnight at 37 0 C.
  • LB Luria Bertani
  • the PCR mixture (in a final volume of 25 ⁇ l) contained 10 ⁇ l of the centrifuged cell lysate, 2.0 ⁇ l dNTPs (10 mM), 2.5 ⁇ l of Taq polymerase buffer, 0.5 ⁇ l of screening primers (to give a final concentration of 100 picomoles) (2 IM 13 FP and ATTBl PCR RP [unique]) and 0.5 ⁇ l of Taq DNA polymerase.
  • the conditions for the screening PCR reaction were: 94 0 C for 2 min, followed by 30 cycles of 94 0 C for 30 s; 58 0 C for 30 s and 72 0 C for 1 min; and a final extension cycle of 72 0 C for 5 minutes and a holding cycle of 4 °C.
  • the PCR products were loaded onto a 1.0 % agarose gel to verify the fragment size.
  • Plasmid DNA 150-200 ng was subjected to DNA sequencing with 2 IM 13 and M13Rev primers using the Applied Biosystems BigDye Terminator V 3.1 Cycle sequencing Kit (Applied Biosystems P/N 4336917) according to the manufacturer's instructions.
  • the primer sequences are shown in Table 4. Sequencing reactions were analyzed on ABI 3100 Genetic Analyzer (Applied Biosystems P/N 628-0030). After sequence confirmation of the insert, pDONR221_INSP216 variant-6HIS was then used to create the expression clones.
  • Plasmid DNA (2 ⁇ l or approx. 150 ng) of pDONR221JNSP216V-6HIS was then used in a recombination reaction containing 1.5 ⁇ l of either pEAK12d vector or pDEST12.2 vector (0.1 ⁇ g / ⁇ l), 2 ⁇ l LR buffer and 1.5 ⁇ l of LR clonase (Invitrogen) in a final volume of 10 ⁇ l.
  • reaction was stopped by addition of 1 ⁇ l proteinase K (2 ⁇ g/ ⁇ l) and incubated at 37
  • DH5D strain (Invitrogen) as follows: a 50 ⁇ l aliquot of DH5D cells was thawed on ice and 2 ⁇ l of reaction mixture added. The mixture was incubated for 30 min on ice and then heat shocked by incubation at 42 0 C for exactly 45 s. Samples were returned to ice and 250 ⁇ l of warm SOC media (room temperature) was added. Samples were incubated with shaking (250 rpm) for 1 h at 37 0 C. The transformation mixture was then plated on Luria Bertani (LB) plates containing Ampicillin (100 ⁇ g/ml) and incubated overnight at 37 °C.
  • LB Luria Bertani
  • the PCR mixture (in a final volume of 25 ⁇ l) contained 10 ⁇ l of the centrifuged cell lysate, 2.0 ⁇ l dNTPs (10 mM), 2.5 ⁇ l of Taq polymerase buffer, 0.5 ⁇ l of screening primers (to give a final concentration of 100 picomoles and 0.5 ⁇ l of Taq DNA polymerase.
  • pEAK12d clones were screened using the primers pEAK12 FP and pEAK12 RP and pDEST12.2 clones were screened using the primers 21M13FP and M13Rev RP.
  • the conditions for the screening PCR reaction were: 94 0 C for 2 min, followed by 30 cycles of 94 °C for 30 s; 60°C for 30 s and 72 °C for 1 min; and a final extension cycle of 72 °C for 5 minutes and a holding cycle of 4 0 C.
  • the PCR products were loaded onto a 1.0 % agarose gel to verify the fragment size.
  • Plasmid mini-prep DNA was prepared from 5 ml cultures using QIAprep Spin Miniprep kit (Qiagen).
  • Plasmid DNA (150 - 200 ng) in the pEAK12d vector was subjected to DNA sequencing with the sequencing primers pEAK12 FP and pEAK12 RP as described above.
  • Plasmid DNA (150 - 200 ng) in the pDEST12.2 vector was subjected to DNA sequencing with the sequencing primers 2 IM 13 FP and M13Rev RP as described above.

Abstract

Cette invention repose sur la découverte que les protéines humaines désignées ici par INSP216, INSP216sv1, INTP216sv2 et INSP216 var sont des protéines contenant un domaine Sushi.
PCT/GB2007/003443 2006-09-15 2007-09-11 Variantes d'épissure fhr-4a WO2008032053A1 (fr)

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GB0618217A GB0618217D0 (en) 2006-09-15 2006-09-15 FHR-4A splicing variants

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997038136A1 (fr) * 1996-04-09 1997-10-16 Bard Diagnostic Sciences, Inc. Methodes et composition de depistage ou de modulation d'un antigene associe aux tumeurs
WO1999018200A1 (fr) * 1997-10-06 1999-04-15 Sagami Chemical Research Center Proteines ressemblant au facteur h du complement humain et adnc codant ces proteines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997038136A1 (fr) * 1996-04-09 1997-10-16 Bard Diagnostic Sciences, Inc. Methodes et composition de depistage ou de modulation d'un antigene associe aux tumeurs
WO1999018200A1 (fr) * 1997-10-06 1999-04-15 Sagami Chemical Research Center Proteines ressemblant au facteur h du complement humain et adnc codant ces proteines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOZSI MIHALY ET AL: "FHR-4A: a new factor H-related protein is encoded by the human FHR-4 gene", EUROPEAN JOURNAL OF HUMAN GENETICS, vol. 13, no. 3, March 2005 (2005-03-01), pages 321 - 329, XP002465547, ISSN: 1018-4813 *
SKERKA C ET AL: "A novel short consensus repeat-containing molecule is related to human complement factor H.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 5 FEB 1993, vol. 268, no. 4, 5 February 1993 (1993-02-05), pages 2904 - 2908, XP002465540, ISSN: 0021-9258 *

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