WO2007072012A1 - Nouveaux membres de la famille kazal des inhibiteurs de serine protease - Google Patents

Nouveaux membres de la famille kazal des inhibiteurs de serine protease Download PDF

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WO2007072012A1
WO2007072012A1 PCT/GB2006/004846 GB2006004846W WO2007072012A1 WO 2007072012 A1 WO2007072012 A1 WO 2007072012A1 GB 2006004846 W GB2006004846 W GB 2006004846W WO 2007072012 A1 WO2007072012 A1 WO 2007072012A1
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seq
polypeptide
disease
nucleic acid
acid molecule
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PCT/GB2006/004846
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Richard Joseph Fagan
David Michalovich
Simon White
Richard Mitter
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Ares Trading S.A.
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Publication of WO2007072012A1 publication Critical patent/WO2007072012A1/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
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor, ovomucoid

Definitions

  • This invention relates to novel proteins, termed INSP 170, INSP222 and INSP223, herein identified as novel human members of the Kazal family of serine protease inhibitors, and to the use of these proteins and nucleic acid sequences from the encoding genes in the diagnosis, prevention and treatment of disease.
  • Serine protease inhibitors are found in animals, plants and microorganisms and are involved in many different biological processes, from blood coagulation in vertebrates to response to mechanical damage in plants. Serine protease inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Serine protease inhibitors can be divided into approximately 20 different families on the basis of sequence and structural similarity (Otlewski, J et al 1999 Acta Biochim Pol; 46 (3): 531-365 and Bode, W and Huber, R 2000 Biochim Biophys Acta 1477(1-2): 241-252, see Table 1).
  • Kunitz family, such as dentrotoxins and bungarotoxins also lack protease inhibitor function and instead have an entirely different activity as K + and Ca 2+ channel blockers.
  • Active serine protease inhibitors operate by strongly binding the active site of their target protease in place of the preferred substrate. About 12 residues of the inhibitor make close contacts with the protease. However, the strength of interaction and specificity of inhibitors depends, in particular, on the primary specificity site "Pl”. (Laskowski Jr. M, Encyclopedia of Molecular Biology, vol 4).
  • inhibitors of trypsin and trypsin- like enzymes tend to have Lys or Arg as the Pl residue
  • inhibitors of chymotrypsin and chymotrypsin-like enzymes tend to have either Pro, Tyr, Phe, Leu or Met as their Pl residue
  • inhibitors of elastase-like enzymes have Ala or Ser as their Pl residue
  • Thr at the Pl position is common in proteinase inhibitors present in secretory proteins.
  • Serine protease inhibitor Three different types can be distinguished on the basis of mechanism: (i) canonical (standard mechanism) inhibitors, (ii) non-canonical inhibitors, and (iii) serpins. (i) Canonical inhibitors are relatively small proteins. They all possess an exposed and convex binding loop of a characteristic canonical conformation, but are otherwise unrelated in structure. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate.
  • Non-canonical inhibitors originating from blood sucking organisms, specifically block enzymes of the blood-clotting cascade.
  • Serpins interact with their target proteases in a substrate-like manner. However, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.
  • the invention is based on the surprising finding that the human INSP 170, INSP222 and INSP223 polypeptides are members of the Kazal family of serine protease inhibitors.
  • INSP 170 and INSP223 are predicted to contain 2 Kazal domains and to show homology to known mammalian carnivore double-headed protease inhibitors ("bikazals").
  • the Kazal family of serine protease inhibitors includes both proteins having only one Kazal domain and proteins having more than one Kazal domain.
  • polypeptide which:
  • (i) comprises the amino acid sequence as recited in SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 and/or SEQ ID NO:28;
  • (ii) is a fragment thereof which is a member of the Kazal family of serine protease inhibitors, or has an antigenic determinant in common with the polypeptides of (i); or
  • the polypeptide according to the first embodiment of the first aspect of the invention comprises the amino acid sequence as recited in SEQ ID NO:28.
  • polypeptide which consists of the amino acid sequence as recited in SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 , SEQ ID NO:26 and/or SEQ ID NO:28.
  • polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as the "INSP222 protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as the "INSP222 protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as the "INSP222 protein sequence exon 1".
  • SEQ ID NO:20 is referred to hereafter as the "INSP222 protein sequence exon 2".
  • the polypeptide having the sequence recited in SEQ ID NO:22 is referred to hereafter as the "INSP222 protein sequence exon 3".
  • the polypeptide having the sequence recited in SEQ ID NO:24 is referred to hereafter as the "INSP222 protein sequence exon 4".
  • the polypeptide having the sequence recited in SEQ ID NO:26 is referred to hereafter as the "INSP222 protein sequence exon 5".
  • the polypeptide having the sequence recited in SEQ ID NO:28 is referred to hereafter as the "INSP222 full protein sequence”.
  • INSP222 polypeptides as used herein includes polypeptides comprising the INSP222 protein sequence exon 1, the INSP222 protein sequence exon 2, the INSP222 protein sequence exon 3, the INSP222 protein sequence exon 4, the INSP222 protein sequence exon 5 and the INSP222 full protein sequence.
  • a polypeptide which:
  • (i) comprises the amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and/or SEQ ID NO: 16;
  • (ii) is a fragment thereof which is a member of the Kazal family of serine protease inhibitors, or has an antigenic determinant in common with the polypeptides of (i); or
  • the polypeptide according to this first embodiment of the first aspect of the invention comprises the amino acid sequence as recited in SEQ ID NO: 12 or SEQ ID NO 16.
  • polypeptide which consists of the amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14 and/or SEQ ID NO: 16.
  • the polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as the "INSP 170 protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO:4 is referred to hereafter as the "INSP170 protein sequence exon 2".
  • the polypeptide having the sequence recited in SEQ ID NO:6 is referred to hereafter as the "INSP 170 protein sequence exon 3 ".
  • the polypeptide having the sequence recited in SEQ ID NO:8 is referred to hereafter as the "INSP 170 protein sequence exon 4".
  • the polypeptide having the sequence recited in SEQ ID NO: 10 is referred to hereafter as the "INSP 170 protein sequence exon 5".
  • the polypeptide having the sequence recited in SEQ ID NO:12 is referred to hereafter as the "INSP170 full protein sequence".
  • the polypeptide having the sequence recited in SEQ ID NO: 14 is referred to hereafter as the "INSP 170 mature protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO: 16 is referred to hereafter as the "INSP 170 full mature protein sequence”.
  • INSP 170 polypeptides as used herein includes polypeptides comprising the INSP 170 protein sequence exon 1, the INSP 170 protein sequence exon 2, the INSP 170 protein sequence exon 3, the INSP170 protein sequence exon 4, the INSP170 protein sequence exon 5, the INSP 170 full protein sequence, the INSP 170 mature protein sequence exon 1 and the INSP 170 full mature protein sequence.
  • amino acid residues 1-18 of the INSP170 full length polypeptide form a signal peptide, as shown in the schematic representation below: MKGFTAFAEL GLAVTAWATS PYGKSGFLSN SHAFMLTVNC SRYIKGLKTA
  • the INSP 170 exon 1 and INSP 170 full length polypeptide sequences without the postulated signal sequence are recited in SEQ ID NO:14 and SEQ ID NO:16, respectively.
  • the polypeptide having the sequence recited in SEQ ID NO: 14 is referred to herein as the "INSP 170 mature protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO: 16 is referred to hereafter as the "INSP 170 full mature protein sequence".
  • the INSP 170 protein sequence exon 1 or the INSP 170 mature protein sequence exon 1 may consist of only part of the sequence recited in SEQ ID NO:2 or SEQ ID NO: 14 respectively. Where this is the case, the INSP 170 protein sequence exon 1 or INSP 170 mature protein sequence exon 1 will differ from that recited in SEQ ID NO:2 or SEQ ID NO: 14 respectively by way of deletion of one or more amino acid residues from the 3' terminal end of the INSP 170 protein sequence exon 1 or INSP 170 mature protein sequence exon 1.
  • the INSP 170 protein sequence exon 1 preferably consists of residues 1-37 as recited in SEQ ID NO:2, it may alternatively consist of residues 1-36, 1- 35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29 or a polypeptide in which further residues have been deleted from the 3' end of SEQ ID NO:2.
  • Polypeptides comprising the sequence of SEQ ID NO:2 with one or more residues deleted from its 3' end in combination with the sequences recited in one or more of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO: 10 are thus specifically included within the scope of the invention.
  • the INSP 170 mature protein sequence exon 1 preferably consists of residues 1-19 as recited in SEQ ID NO:14, it may alternatively consist of residues 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11 or a polypeptide in which further residues have been deleted from the 3' end of SEQ ID NO:14.
  • Polypeptides comprising the sequence of SEQ ID NO:14 with one or more residues deleted from its 3' end in combination with the sequences recited in one or more of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO: 10 are thus specifically included within the scope of the invention.
  • polypeptide which: (i) comprises the amino acid sequence as recited in 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 and/or SEQ ID NO:46;
  • (ii) is a fragment thereof which is a member of the Kazal family of serine protease inhibitors, or has an antigenic determinant in common with the polypeptides of (i); or
  • the polypeptide according to this first embodiment of the first aspect of the invention comprises the amino acid sequence as recited in SEQ ID NO:42 or SEQ ID NO 46.
  • a polypeptide which consists of the amino acid sequence as recited in 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 and/or SEQ ID NO:46.
  • polypeptide having the sequence recited in SEQ ID NO:30 is referred to hereafter as the "INSP223 protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO:30 is referred to hereafter as the "INSP223 protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO:30 is referred to hereafter as the "INSP223 protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO:30 is referred to hereafter as the "INSP223 protein sequence exon 1".
  • SEQ ID NO:32 is referred to hereafter as the "INSP223 protein sequence exon 2".
  • the polypeptide having the sequence recited in SEQ ID NO:34 is referred to hereafter as the "INSP223 protein sequence exon 3 ".
  • the polypeptide having the sequence recited in SEQ ID NO:36 is referred to hereafter as the "INSP223 protein sequence exon 4".
  • the polypeptide having the sequence recited in SEQ ID NO:38 is referred to hereafter as the "INSP223 protein sequence exon 5".
  • the polypeptide having the sequence recited in SEQ ID NO:40 is referred to hereafter as "INSP223 protein sequence exon 6".
  • the polypeptide having the sequence recited in SEQ ID NO:42 is referred to hereafter as the "INSP223 full protein sequence".
  • the polypeptide having the sequence recited in SEQ ID NO:44 is referred to hereafter as the "INSP223 mature protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO:46 is referred to hereafter as the "INSP223 full mature protein sequence”.
  • INSP223 polypeptides as used herein includes polypeptides comprising the INSP223 protein sequence exon 1, the INSP223 protein sequence exon 2, the INSP223 protein sequence exon 3, the INSP223 protein sequence exon 4, the INSP223 protein sequence exon 5, the INSP223 protein sequence exon 6, the INSP223 full protein sequence, the INSP223 mature protein sequence exon 1 and the INSP223 full mature protein sequence.
  • amino acid residues 1-18 of the INSP223 full length polypeptide form a signal peptide, as shown in the schematic representation below:
  • the INSP223 exon 1 and INSP223 full length polypeptide sequences without the postulated signal sequence are recited in SEQ ID NO:44 and SEQ ID NO:46, respectively.
  • the polypeptide having the sequence recited in SEQ ID NO:44 is referred to herein as the "INSP223 mature protein sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO:46 is referred to hereafter as the "INSP223 full mature protein sequence".
  • Kazal family of serine protease inhibitors refers to a molecule containing at least one Kazal domain. Kazal domains are found in many proteins with diverse roles. In particular, Kazal domains are found in many members of the canonical (standard mechanism) family of serine protease inhibitors such as pancreatic secretory trypsin inhibitor, avian ovomucoid, acrosin inhibitor and elastase inhibitor. Between 1 and 7 Kazal domains are found in each of these proteins.
  • Kazal domains are also seen in other proteins such as agrin and follistatin which are not known to function as serine protease inhibitors.
  • Further examples of Kazal domain-containing proteins are SPINKl, SPINK2, SPINK5, recombinant TgPI-I, LDTI, RECK, Agrin and Follistatin. Each of these is discussed briefly below: SPINKl is a pancreatic secretory trypsin inhibitor. It is believed to prevent the premature activation of pancreatic zymogens by trypsin, otherwise resulting in pancreatitis. It is also expressed by the mucosa of the GI tract where it's thought to prevent the breakdown of the mucosal cells (Witt et al, 2000, Nature: Genetics, 25, 213-216; OMIM: 167790).
  • SPINK2 is an acrosin-trypsin inhibitor expressed in testis, epididymis and seminal vesicle (OMIM: 605753). Acrosin is the major protease of mammalian spermatozoa. SPINK2 is a strong inhibitor of acrosin in both the male and female genital tracts.
  • SPINK5 has been shown to have an in-vitro anti-trypsin activity and is expressed in epithelia and in the thymus (OMIM: 605010).
  • the intracellular parasite Toxoplasma gondii secretes protease inhibitors that protect the parasite from proteolytic enzymes found in the lower intestine, (e.g. Recombinant TgPI-I inhibits trypsin, chymotrypsin, pancreatic elastase and neutrophil elastase) (Morris et al, 2002, Biological Chemistry, 277, 45259-45266).
  • protease inhibitors that protect the parasite from proteolytic enzymes found in the lower intestine, (e.g. Recombinant TgPI-I inhibits trypsin, chymotrypsin, pancreatic elastase and neutrophil elastase) (Morris et al, 2002, Biological Chemistry, 277, 45259-45266).
  • LDTI a secreted Kazal domain-containing protein isolated from the medicinal leech has been shown to inhibit human mast cell tryptase.
  • the physiological role of tryptase are not clear though the central role of mast cells in allergic and inflammatory reactions suggests that tryptase may be involved in the pathogenesis of disorders such as asthma, interstitial lung disease, psoriasis, arthritis, gingivitis and peridontitis (Stubbs et al, 1997, JBC, 272, 19931-19937).
  • RECK inhibits MMP9, MMP2 and MMP 14. The precise mechanism of inhibition is not known and may not be due to direct interaction between the MMPs and RECK's Kazal domains. RECK down-regulation by oncogenic signals may facilitate tumor invasion and metastasis.
  • Agrin is a neuronal aggregating factor that induces the aggregation of acetylcholine receptors and other postsynaptic proteins on muscle fibers and is crucial for the formation of the neuromuscular junction. Despite containing Kazal domains, Agrin is not known to function as a serine protease inhibitor.
  • Follistatin regulates several signaling pathways in a cell and tissue specific manner by inactivating TGF ⁇ -like growth factors.
  • Follistatin is an antagonist of activin and is also a specific inhibitor of the secretion and biosynthesis of follicle-stimulating hormone (FSH) (Innis et al, 2003, JBC, 278, 39969-39977).
  • FSH follicle-stimulating hormone
  • Kazal-type trypsin inhibitors are synthesized in follicle cells, secreted, accumulated in the egg chorion during oocyte development and released at spawning to activate spermatozoa motility (Oda, S. et al., (1998), Dev. Biol., 1:204(1): 55-63).
  • spermatozoa motility Oda, S. et al., (1998), Dev. Biol., 1:204(1): 55-63.
  • Known human members of the Kazal family of serine protease inhibitors, such as SPINKl and SPINK4 are typically short (-80 amino acids) and are composed of an N-terminal signal peptide followed by a C-terminal Kazal domain.
  • Kazal domains generally consist of 50 to 60 amino acid residues, although a protein having a single domain of only 36 residues has recently been described (Nirmala, X. et al. Eur. J. Biochem. (2001) 268, 2064-2073.
  • Kazal domain includes 2 short ⁇ -helices and a 3 -stranded anti-parallel ⁇ - sheet. These structures form 3 loops (A, B and C) that are stabilised and closed into rings by disulfide bonds between cysteine residues (Laskowski, M. & Kato, I. (1980) Protein inhibitors of proteinases. Ann. Rev. Biochem. 49, 593-626). Typically, Kazal domains contain six cysteine residues. The A-ring is closed by a disulfide bond between Cysl- CysV and the A- and B-rings are separated by the CysII-CysIV bridge.
  • the CysI-CysV bridge and CysII-CysIV bridge also serve to link the N-terminus and the reactive site region, respectively, to the central ⁇ -helix.
  • the C-ring is closed by a disulfide bridge between CysIII-CysVI, which links the C-terminus to the central ⁇ -sheet.
  • the number of amino acid residues in the A-loop between Cysl and CysII varies from 18 in avian ovomucoids to just one in the insect inhibitors. Generally, the C-loop is between 31 and 34 residues.
  • bdellin-B3 from leech contains a C-loop of just 27 residues (Fritz, H. & Krejcu, K. (1976) Trypsin-plasmid inhibitors (bdellins) from leeches. In Methods in Enzymology (L. Lorand, ed.), 45, pp. 797-806. Academic Press, New York, USA).
  • Arg at P 1 has been shown to confer specificity for inhibition of trypsin whilst preventing interaction with trypsinogen.
  • the presence of a Met or Tyr at P 1 has been shown to confer specificity for chymotrypsin-subilisin-elastase (Laskowski & Kato, Ann. Rev. Biochem., (1980) 49:593-626).
  • Many serine protease inhibitors play a role in disease processes.
  • Inhibitors can be engineered to be specific for some serine proteases only - similar enzymes involved in different contexts can remain unaffected. Unique structural features have been identified in some naturally occurring serine protease inhibitors and a range of pentapeptide analogues that incorporated these features have been synthesized. These analogues have a range of selective actions against trypsin, chymotrypsin and kallikrein.
  • the "Kazal family of serine protease inhibitors” may be a molecule containing a Kazal domain detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.000001 or 0.0000001.
  • the "Kazal family of serine protease inhibitors” 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, 0.000001 or 0.0000001.
  • a polypeptide according to any of the above-described aspects of the invention acts as a member of the Kazal family of serine protease inhibitors.
  • act as a member of the Kazal family of serine protease inhibitors we refer to polypeptides that comprise a Kazal domain and exhibit serine protease inhibitor activity.
  • polypeptides that comprise amino acid sequence or structural features that can be identified as conserved features within the polypeptides of this family.
  • N-terminal Kazal domain of the INSP 170 and INSP223 polypeptides of the present invention show specificity for trypsin since the Pl residue is arginine.
  • Kazal domain of the INSP 170, INSP222 and INSP223 polypeptides of the present invention show specificity for chymotrypsin or chymotrypsin-like enzymes since the C- terminal Kazal domain contains a methionine at the Pl position (Nirmala, X. et al., Eur. J.
  • An example of a method in which the activity of a polypeptide according to the present invention can be determined is by measuring inhibition of a target serine protease.
  • trypsin is used as the target enzyme of the polypeptide according to the present invention
  • its inhibition can be measured with the azocoll assay (Chavira, RJ. et al., (1984) Assaying proteinases with azocoll. Anal. Biochem., 136, 446-450).
  • This assay can be modified for microplate ELISA photometric readers (see Wedde, M. et al. (1998), Eur. J. Biochem., 255, 535-543).
  • the polypeptides of the first aspect of the invention may further comprise a histidine tag.
  • the histidine tag is found at the C-terrninal of the polypeptide.
  • the histidine tags comprises 1-10 histidine residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably the histidine tag comprises 6 histidine residues.
  • an "antigenic determinant" of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR).
  • TCR T-cell receptor
  • 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
  • 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.
  • polypeptides of the present invention may modulate a variety of physiological and pathological processes or disorders.
  • the biological activity or function of these polypeptides can be examined in systems that allow the study of such modulatory activities, using a variety of suitable assays.
  • the 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 molecules are specifically excluded from the scope of the invention.
  • genomic DNA larger than 10 kbp (kilo baise 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 nucleic acid molecule comprises comprises the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP170 protein sequence exon 1), SEQ ID NO:3 (encoding the INSP170 protein sequence exon 2), SEQ ID NO:5 (encoding the INSP170 protein sequence exon 3), SEQ ID NO:7 (encoding the INSP170 protein sequence exon 4), SEQ ID NO:9 (encoding the INSP 170 protein sequence exon 5), SEQ ID NO:11 (encoding the INSP170 full protein sequence), SEQ ID NO:13 (encoding the INSP170 mature protein sequence exon 1), SEQ ID NO: 15 (encoding the INSP 170 full mature protein sequence), SEQ ID NO: 17 (encoding the INSP222 protein sequence exon 1), SEQ ID NO: 19 (encoding the INSP222 protein sequence exon 2), SEQ ID NO:21 (encoding the INSP222 protein sequence exon 3), SEQ ID NO:23 (encoding the INSP222 protein sequence exon
  • the invention further provides that the purified nucleic acid molecule consists of the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP 170 protein sequence exon 1), SEQ ID NO:3 (encoding the INSP170 protein sequence exon 2), SEQ ID NO:5 (encoding the INSP170 protein sequence exon 3), SEQ ID NO:7 (encoding the INSP170 protein sequence exon 4), SEQ ID NO:9 (encoding the INSP 170 protein sequence exon 5), SEQ ID NO: 11 (encoding the INSP 170 full protein sequence), SEQ ID NO: 13 (encoding the INSP 170 mature protein sequence exon 1), SEQ ID NO: 15 (encoding the INSP 170 full mature protein sequence), SEQ ID NO: 17 (encoding the INSP222 protein sequence exon 1), SEQ ID NO: 19 (encoding the INSP222 protein sequence exon 2), SEQ ID NO:21 (encoding the INSP222 protein sequence exon 3), SEQ ID NO:23 (encoding the IN
  • the purified nucleic acid according to the above-described aspect of the invention is a nucleic acid having the sequence of SEQ ID NO: 11 (encoding the INSP 170 full length protein sequence), SEQ ID NO: 15 (encoding the INSP 170 mature full length protein sequence), SEQ ID NO:27 (encoding the INSP222 full protein sequence), SEQ ID NO:41 (encoding the INSP223 full protein sequence) or SEQ ID NO:45 (encoding the INSP223 full mature protein sequence).
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/rnl 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 a member of the Kazal family of serine protease inhibitors of the first aspect of the invention.
  • the ligand inhibits the function of a polypeptide of the first aspect of the invention which is a member of the Kazal family of serine protease inhibitors.
  • Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Such compounds may be identified using the assays and screening methods disclosed herein.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification of the function of the INSP 170, INSP222 and INSP223 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease.
  • the term "disease” also includes disorders.
  • Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.
  • Another aspect of this invention resides in the use of an INSP 170, INSP222 or INSP223 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against Kazal domain related disorders.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of Kazal domain related disorders, comprising determining the ability of a compound to bind an INSP 170, INSP222 or INSP223 gene or polypeptide, or a fragment thereof.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of Kazal domain related disorders, comprising testing for modulation of the activity of an INSP 170, INSP222 or INSP223 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 members of the Kazal family of serine protease inhibitors are implicated "Kazal domain related disorder".
  • Such diseases may include cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours, myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis 1 sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions.
  • such diseases include chronic inflammatory diseases such as chronic pancreatitis, including idiopathic, hereditary and alcoholic chronic pancreatitis, fibrocalculous pancreatic diabetes, asthma, interstitial lung disease, psoriasis, arthritis gingivitis, peridontitis, cancers such as genitourinary tumours including ovarian cancer, prostate cancer and also breast cancer and esophageal cancer, Netherton Syndrome, fertility disorders including male (infertility and female (infertility, arthritis, Alzheimer's disease and transmissible prion encephalopathies, liver disease including hepatitis, juvenile cirrhosis and hepatocellular carcinoma, cardiovascular disease, hereditary thrombophilia, bleeding, coronary atherosclerosis, increased risk or coronary heart disease, myocardial infarction, hereditary angioedema, acquired angioedema, hereditary angioneurotic edema, capillary leak syndrome, essential hypertension, pregnancy-induced
  • the diseases are those in which members of the Kazal family of serine protease inhibitors are implicated.
  • the eighth aspect of the invention there is also provided 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 contraception.
  • These moieties of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention may also be used in the manufacture of a medicament for the treatment of such diseases or for contraception.
  • embodiments of this invention provide for the use of the polypeptides in inflammatory diseases, autoimmue diseases, encephalopathies, cerebrovascular/cardiovascular diseases, blood clotting disorders, cancer, lung diseases, liver diseases or male infertility.
  • the encephalopathy is selected from dementia, Alzheimer's disease or transmissible prion encephalopaty
  • the cerebrovascular disease is selected from cerebral ischemia/reperfusion or stroke
  • the cardiovascular disease is selected from thrombosis, hereditary thrombophilia, bleeding, essential hypertension, pregnancy-induced hypertension (preeclampsia), coronary atherosclerosis, coronary heart disease (CHD) or myocardial infarction (MI).
  • the lung disease is selected from cystic fibrosis, adult respiratory distress syndrome, COPD, asthma or emphysema.
  • the blood clotting disorder is selected from disorders affecting medium sized arteries, intracranial aneurysms, cervicocephalic arterial dissections or fibromuscular dysplasia.
  • the liver disease is selected from hepatitis, juvenile cirrhosis or hepatocellular carcinoma.
  • the inflammatory disease is selected from angioedema, allergenic hypersensitivity, inflammatory skin diseases or chronic inflammatory diseases.
  • the angioedema is selected from heriditary angioedema, acquired angioedema or hereditary angioneurotic edema.
  • the inflammatory skin disease is selected from atopic dermatitis or psoriasis hi one embodiment, cancer is selected from breast cancer or prostate cancer.
  • the autoimmune disease is selected from rheumatoid arthritis, Crohn's disease, Multiple Sclerosis, inflammatory bowel disease (IBD), systemic lupus erithematosus (SLE), Sjogren's syndrome or myasthenia gravis.
  • IBD inflammatory bowel disease
  • SLE systemic lupus erithematosus
  • Sjogren's syndrome myasthenia gravis.
  • 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 prostate cancer and male (infertility disorders.
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • a number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient.
  • the invention also provides kits that are useful in these methods for diagnosing disease.
  • the disease diagnosed by a method of the ninth aspect of the invention is a disease in which members of the Kazal family of serine protease inhibitors are implicated, as described above.
  • the invention provides for the use of a polypeptide of the first aspect of the invention as a Kazal family of serine protease inhibitors.
  • Suitable uses of the polypeptides of the invention as Kazal family of serine protease inhibitors include screening methods to identify ligands and other agonist or antagonist molecules that are useful in therapy and diagnosis of diseases and conditions in which this category of protein is implicated.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically- acceptable carrier.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, including, but not limited to cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours, myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation and organ transplant rejection; cardiovascular disorders,
  • the disease is a disease in which members of the Kazal family of serine protease inhibitors are implicated, as described above.
  • 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 disease is a disease in which members of the Kazal family of serine protease inhibitors are implicated, as described above.
  • 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 INSP 170, INSP222 and INSP223 polypeptides are Kazal domain containing proteins and thus have roles in many disease states. Antagonists of the INSP 170, INSP222 and INSP223 polypeptides are of particular interest as they provide a way of modulating these disease states.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be used in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • “functional equivalent” refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention.
  • a functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional equivalent” is intended to include the fragments, mutants, hybrids, variants, analogues, 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 INSP 170, INSP222 or INSP223 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 INSP 170, INSP222 or INSP223 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 INSP 170, INSP222 or INSP223 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 polypeptides that reflect their identity as novel members of the Kazal family of serine protease inhibitors.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide, hi such polypeptides, 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.
  • polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • a polypeptide of the invention that may comprise a sequence having at least 85% of homology with INSP 170, INSP222 or INSP223 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 with INSP 170, INSP222 or INSP223 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 INSP 170, INSP222 or INSP223 polypeptide.
  • heterologous sequences that can be comprised in the fusion proteins either at the N- or C-terminus, include: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc regions), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, and sequences allowing purification by affinity chromatography.
  • Fc regions immunoglobulin constant regions
  • heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, 2003, Appl Microbiol Biotechnol, 60:523-33).
  • additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by the a stretch of Histidines forming the so-called "histidine tag" (Gentz et al.
  • the heterologous sequence can be eliminated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease.
  • the INSP 170, INSP222 or INSP223 polypeptides may be purified by means of a hexa-histidine peptide fused at the C-terminus of INSP 170, INSP222 or INSP223.
  • 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:47) introduced between the sequence of the substances of the invention and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids (i.e. an increased half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • the protein is fused to the constant region of an Ig molecule.
  • IgGl immunoglobulin G
  • IgG2 or IgG4 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.
  • polypeptides 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.
  • the 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 INSP 170, INSP222 and INSP223 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 INSP 170, INSP222 and INSP223 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
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • any substitution should be preferably a "conservative” or “safe” substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties (e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.
  • non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the PMP- 22/EMP/MP20/Claudin-like molecule 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 3.
  • a non-exhaustive list of amino acid derivatives also include aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydro- isoquinoline-3-COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidine-4- carboxylic acid, L-homoproline, 3,4-dehydro- ⁇ roline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine, and phenylglycine.
  • amino acid derivative is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids.
  • the amino acid derivative may contain substituted or non-substituted, linear, branched, or cyclic alkyl moieties, and may include one or more heteroatoms.
  • the amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem- Novabiochem AG, Switzerland; Bachem, USA).
  • polypeptides of the first aspect of the invention have a degree of sequence identity with the INSP 167 or INSP 170 polypeptides, or with active fragments thereof, of greater than 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the BiopendiumTM search database may be used.
  • INSP222 and INSP223 polypeptide sequences.
  • significant structural homology is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.
  • polypeptides of the first aspect of the invention also include fragments of the INSP 170, INSP222 and INSP223 polypeptides and fragments of the functional equivalents of the INSP 170, INSP222 and INSP223 polypeptides, provided that those fragments are members of the Kazal family of serine protease inhibitors or have an antigenic determinant in common with the INSP 170, INSP222 or INSP223 polypeptides.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP 170, INSP222 and INSP223 polypeptides or one of their 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, preferably 7-20, more preferably 10-15 amino acids in length.
  • Nucleic acids according to the invention are preferably 5-400 nucleotides in length, preferably 10-300 nucleotides, preferably 15-250, preferably 20-200, preferably 30-150, preferably 50-10 nucleotides in length.
  • Polypeptides according to the invention are preferably 5-150 amino acids in length, preferably 10-100, preferably 15-75, preferably 25-50 amino acids in length.
  • Fragments of the full length INSP 170, INSP222 and INSP2223 polypeptides may consist of combinations of 2, 3, 4 or all 5 neighbouring exon sequences in the INSP 170 polypeptide sequence, or 2, 3, 4 or all 5 neighbouring exon sequences in the INSP222 polypeptide sequence, or 2, 3, 4, 5 or all 6 neighbouring exon sequences in the INSP223 polypeptide sequence.
  • 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 known members of the Kazal family of serine protease inhibitors.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, 10 4 - fold, 10 5 -fold, 10 6 -fold or greater for a polypeptide of the invention than for known members of the Kazal family of serine protease inhibitors.
  • 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)).
  • 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
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
  • the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode a polypeptide sequence as recited in SEQ ID NO.2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO.12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO.30 SEQ ID NO:32 SEQ ID NO:34 SEQ ID NO:36 SEQ ID NO:38 SEQ ID NO:40 SEQ ID NO:42 SEQ ID NO:44 and/or SEQ ID NO:46 and functionally equivalent polypeptides.
  • 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.
  • the term "nucleic acid molecule” also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al (1993) Anticancer Drug Des. 8:53-63).
  • a nucleic acid molecule which encodes a polypeptide of this invention may be identical to the coding sequence of one or more of the nucleic acid molecules disclosed herein.
  • These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ DD NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ DD NO: 18, SEQ DD NO:20, SEQ DD NO:22, SEQ DD NO:24, SEQ DD NO:26, SEQ DD NO:28, SEQ DD NO:30 SEQ DD NO:32 SEQ DD NO:34 SEQ DD NO:36 SEQ DD NO:38 SEQ DD NO:40 SEQ DD NO:42 SEQ DD NO:44 and/or SEQ DD NO:46.
  • nucleic acid molecules may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non- translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
  • the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
  • nucleic acid molecules of the second and third aspects of the invention may also encode the 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 splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules
  • antisense molecules such as oligonucleotides
  • oligonucleotides can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56,
  • 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 SX. 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 0 C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INSP 170, INSP222 or INSP223 polypeptides and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to such coding sequences, or is a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98%, 99% or more identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSP 170, INSP222 or INSP223 polypeptides.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INSP 167 or INSP 170 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the genes 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 INSP 170, INSP222 or INSP223 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:11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ E) NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ E) NO:27, SEQ E) NO:29, SEQ E) NO:31, SEQ E) NO:33, SEQ E) NO:35, SEQ TD NO:37, SEQ E) NO:39, SEQ E) NO:41, SEQ E) NO:43 or SEQ E) NO:45) are particularly useful probes.
  • Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • telomere shortening uses universal primers to retrieve unknown nucleic acid sequence adjacent a known locus.
  • Inverse PCR may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
  • capture PCR involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119).
  • Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • nucleic acid molecules of the present invention may be used for chromosome localisation. In this technique, a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • mapping of relevant sequences to chromosomes is an important step in the confirmatory correlation of those sequences with the gene-associated disease.
  • the physical position of the sequence on the chromosome can be correlated with genetic map data.
  • genetic map 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.
  • 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.
  • Gene silencing approaches may also be undertaken to down-regulate endogenous expression of a gene encoding a polypeptide of the invention.
  • 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.
  • 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.
  • 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
  • the vectors pCR4-TOPO-INSP187-EC-SV2, pENTR_INSP187ECSV2-6HIS, pEAK12d_INSP187ECSV2-HIS and pDEAT12.2_INSP187ECSV2-6HIS are preferred examples of suitable vectors for use in accordance with the aspects of this invention relating to INSP222.
  • 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 Sambrook et al, ⁇ supra). Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs 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 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.
  • 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.
  • vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding-sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • stable expression is preferred.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene.
  • Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
  • Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.
  • yeast cells for example, S. cerevisiae
  • Aspergillus cells examples include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
  • any number of selection systems are 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 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk " or aprt* cells, respectively.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. MoI. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • DHFR dihydrofolate reductase
  • methotrexate methotrexate
  • npt which confers resistance to the aminoglycosides neomycin and G-418
  • als or pat which confer resistance to chlorsulfuron and phosphinotricin acety
  • 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.
  • polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • FACS fluorescence activated cell sorting
  • the 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 INSP 170, INSP222 or INSP223 polypeptide as a target for screening candidate drugs for treating or preventing Kazal domain related disorders.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with an INSP 170, INSP222 or INSP223 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 an INSP 170, INSP222 or INSP223 polypeptide with a candidate compound, and selecting compounds that bind said INSP 170, INSP222 or INSP223 polypeptide at the surface of said cells and/or that modulate the activity of the INSP 170, INSP222 or INSP223 polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound having Kazal domain activity, and further preferably a compound that can be used for treating INSP 170, INSP222 or INSP223 related disorders, or as a lead to develop drugs for treating Kazal domain related disorders.
  • a “biologically active” compound preferably is a compound that modulates the activity of INSP 170, INSP222 or INSP223.
  • 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 Kazal domain related disorder, such as an animal model.
  • Preferred selected compounds are agonists of INSP 170, INSP222 or INSP2230, i.e., compounds that can bind to INSP 170, INSP222 or INSP223 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 INSP 170, INSP222 or INSP223 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INSP 170, INSP222 or INSP223 polypeptide.
  • a further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INSP 170,
  • INSP222 or INSP223 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INSP 170, INSP222 or INSP223 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 INSP 170, INSP222 or INSP223 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 Kazal domain related disorder in a subject.
  • the determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc.
  • the polypeptides may be used in essentially pure form, in suspension, immobilised 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 INSP 170, INSP222 or INSP223 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 an INSP 170, INSP222 or INSP223 polypeptide) or any cell that expresses an endogenous INSP 170, INSP222 or INSP223 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.).
  • prokaryotic cells such as bacteria
  • eukaryotic cells such as yeast cells, mammalian cells, insect cells, plant cells, etc.
  • yeast 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.
  • primary or established mammalian cell cultures e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a particular example is cotransfecting a construct expressing a polypeptide according to the invention, or a fragment such as the LBD, in fusion with the GAL4 DNA binding domain, into a cell together with a reporter plasmid, an example of which is pFR-Luc (Stratagene Europe, Amsterdam, The Netherlands).
  • This particular plasmid contains a synthetic promoter with five tandem repeats of GAL4 binding sites that control the expression of the luciferase gene. When a potential ligand is added to the cells, it will bind the GAL4-polypeptide fusion and induce transcription of the luciferase gene.
  • the level of the luciferase expression can be monitored by its activity using a luminescence reader (see, for example, Lehman et al. JBC 270, 12953, 1995; Pawar et al. JBC, 277, 39243, 2002).
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises: (a) contacting a labelled or unlabeled compound with the polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself; or
  • a method such as FRET detection of a 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 an agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which have a polypeptide of the invention on the surface thereof, or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide.
  • a compound capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist.
  • the ligand is labelled.
  • a method of screening for a polypeptide antagonist or agonist compound comprises the steps of:
  • step (c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium; (d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c); and
  • step (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • step (c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium; (d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c); and
  • 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.
  • the INSP 170, INSP222 and INSP223 polypeptides of the present invention may modulate cellular growth and differentiation.
  • the biological activity of the INSP 170, INSP222 and INSP223 exon polypeptides and the INSP170, INSP222 and INSP223 polypeptides can be examined in systems that allow the study of cellular growth and differentiation such as in vitro tissue culture. Stimulation or inhibition of cellular proliferation may be measured by a variety of assays.
  • a solid or liquid medium For example, for observing cell growth inhibition, one can use a solid or liquid medium. In a solid medium, cells undergoing growth inhibition can easily be selected from the subject cell group by comparing the sizes of colonies formed. In a liquid medium, growth inhibition can be screened by measuring culture medium turbity or incorporation of labelled thymidine in DNA. Typically, the incorporation of a nucleoside analog into newly synthesised DNA may be employed to measure proliferation (i. e., active cell growth) in a population of cells. For example, bromodeoxyuridine (BrdU) can be employed as a DNA labelling reagent and anti-BrdU mouse monoclonal antibodies can be employed as a detection reagent.
  • bromodeoxyuridine BrdU
  • anti-BrdU mouse monoclonal antibodies can be employed as a detection reagent.
  • This antibody binds only to cells containing DNA which has incorporated bromodeoxyuridine.
  • detection methods may be used in conjunction with this assay including immunofluorescence, immunohistochemical, ELISA, and colorimetric methods.
  • Kits that include bromodeoxyuridine (BrdU) and anti-BrdU mouse monoclonal antibody are commercially available from Boehringer Mannheim (Indianapolis, IN).
  • the effect of the polypeptide upon cellular differentiation can be measured by contacting stem cells or embryonic cells with various amounts of the INSP 170, INSP222 or INSP223 exon polypeptides and the INSP 170, INSP222 or INSP223 polypeptides and observing the effect upon differentiation of the stem cells or embryonic cells. Tissue-specific antibodies and microscopy may be used to identify the resulting cells.
  • the INSP 170, INSP222 or INSP223 exon polypeptides and the INSP 170, INSP222 or INSP223 polypeptides may also be found to modulate immune and/or nervous system cell proliferation and differentiation in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the INSP 170, INSP222 or INSP223 exon polypeptides and the INSP 170, INSP222 and INSP223 polypeptides include polypeptides that exhibit any of the same growth and differentiation regulating 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 INSP 170, INSP222 or INSP223 exon polypeptides and the INSP170, INSP222 or INSP223 polypeptides, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the INSP 170, INSP222 or INSP223 exon polypeptides and the INSP 170, INSP222 or INSP223 polypeptides.
  • 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.
  • Screening assays known in the art that are suitable for the identification of modulators of the polypeptides of this invention include the GAL4 assay (for example, Jurgen M. Lehmann et al, J. Biol. Chem., Jun 1995; 270: 12953- 12956; Raivio T. et al, J. Clin. Endocrinol. Metab. 86 (2001) 1539- 44), biophysical techniques such as surface plasmon resonance (supplied by Biacore AB, Uppsala, Sweden) and assays based on fluorescence polarisation or FRET for the ligand-induced binding of the co-activator peptide (Mukherjee R. et al, J Steroid Biochem. MoI. Biol. 2002 Jul;81(3):217-25).
  • GAL4 assay for example, Jurgen M. Lehmann et al, J. Biol. Chem., Jun 1995; 270: 12953- 12956; Raivio T.
  • 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). In 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 INSP 170, INSP222 or INSP223 polypeptide or fragment thereof, whereby the fragment is preferably a INSP 170, INSP222 or INSP223 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP 170, INSP222 or INSP223 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;
  • a single chain e.g. scFv
  • single domain antibody or
  • an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • 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 bispeciftc 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 bispeciftc 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
  • 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% or even 99% by weight.
  • compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg.
  • Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • the pharmaceutical compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • an inhibitor compound as described above
  • a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • antagonists are antibodies.
  • such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered.
  • the polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered.
  • Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • 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 mRNA by preventing the transcript from binding to ribosomes.
  • Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. 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 (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • jet injection may also be useful in the formulation of vaccine compositions.
  • a number of suitable methods for vaccination and vaccine delivery systems are described in International patent application WO00/29428.
  • 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.
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a)contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b)contacting a control sample with said probe under the same conditions used in step a); c)and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a)obtaining a tissue sample from a patient being tested for a disease; b)isolating a nucleic acid molecule according to the invention from said tissue sample; and c)diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included.
  • Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • Such diagnostics are particularly useful for prenatal and even neonatal testing.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et ah, Genomics, 5, 874-879 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et 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).
  • FISH Fluorescence in situ hybridization
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996), VoI 274, pp 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al (1996) Proc. Natl. Acad. Sci. 93: 10614-10619).
  • Oligonucleotide pairs may range from two to over one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/25116 (Baldeschweiler et at).
  • 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) a nucleic acid molecule of the present invention
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease in which members of the Kazal family of serine protease inhibitors are implicated, as described above.
  • Figure 1 Top ten results from BLAST against NCBI non-redundant database using SEQ ID NO:26 (INSP 170 polypeptide sequence).
  • Figure 2 Alignment generated by BLAST between SEQ ID NO:26 and the top hit, (P08480) double-headed protease inhibitor, submandibular gland, from cat.
  • FIG. 3 Alignment of the INSP 170 polypeptide sequence (SEQ ID NO:26) to secreted double-headed (“Bikazal”) Kazal domain-containing protein family members.
  • Figure 4 NCBI-CDD analysis of INSP 170
  • Figure 5 Signal P analysis of INSP 170.
  • Figure 6 INSP222 DNA sequence. The position and sense of the PCR primers are indicated by arrows.
  • Figure 7 The nucleotide sequence with translation of the INSP222 PCR product cloned using INSP170fwd and INSP170rev PCR primers.
  • Figure 8 ClustalW alignment of the nucleotide sequence of the INSP222 cloned sequence versus INSP222 predicted sequence.
  • Figure 9 Multiple sequence alignment of INSP 170, INSP221 and INSP222 full length polypeptides. Sequence features are highlighted.
  • Figure 10 Exon Structure of INSP170, INSP221 and INSP222
  • Figure 11 Expression reports for Exon 1 of INSP 170.
  • Figure 12 Expression reports for Exon 2 of INSP 170.
  • Figurel3 Expression reports for Exon 3 of INSP 170.
  • Figure 14 Expression reports for Exon 4 of INSP 170.
  • Figure 15 Expression reports for Exon 5 of INSP 170.
  • Example 1 INSP 170 Protein BLAST Results
  • the top ten hits are shown in Figure 1.
  • the top hit is to a double-headed protease inhibitor found in the submandibular gland of cat.
  • the alignment of INSP 170 with this top hit is shown in Figure 2.
  • INSP 170 has 59% identity over its 143 residues to the top hit.
  • the top hit has a highly significant expectation value of 1 e "32 .
  • the fact that all the top ten hits are from members of the Kazal family of serine protease inhibitors having two Kazal domains (i.e.
  • INSP 170 SEQ ID NO: 12
  • ESfSP 170 shows strong homology to known double-headed (“bikazal") Kazal domain-containing secreted proteins.
  • the six structurally significant cysteine residues found in Kazal domains are also present in INSP 170 and are highlighted by asterisks in Figure 3.
  • the residues highlighted by '#' represent the Pl residues from the reactive sites of each Kazal domain. As mentioned above, Pl residues are believed to confer proteinase inhibitor specificity.
  • the N-terminal Kazal domain of INSP 170 contains an arginine at the Pl position, suggesting that this domain inhibits trypsin and trypsin-like enzymes.
  • the C-terminal Kazal domain of INSP 170 contains a methionine at the Pl position, suggesting that this domain inhibits chymotrypsin and chymotrypsin-like enzymes, (Nirmala, et al 2001).
  • the INSP 170 polypeptide sequence (SEQ ID NO: 12) was analysed against the NCBI conserveed Domain Database http://www.ncbi.nih.gov/Structure/cdd/cdd.shtml. The results are shown in Figure 4. Analysis against the Smart, Pfam, CDD and SPAN collections all returned matches to proteins having Kazal domains with significant E-values. This supports the finding that the INSP 170 polypeptide also contains two Kazal domains and is thus a double-headed member of the Kazal family of serine protease inhibitors.
  • the majority of the assays focus on the major immune system cell types, namely T and B lymphocytes, macrophages and neutrophils. Further assays concentrate on the biology of microglial cells.
  • Assays targeting endothelial cell responses use cultures of primary endothelial cells such as human embryonic umbilical endothelial cells (HUVEC).
  • primary endothelial cells such as human embryonic umbilical endothelial cells (HUVEC).
  • T cell apoptosis is induced by stimulating Jurkat cells (a human T cell line) with recombinant 6 Histidine-tagged Fas Ligand combined with a monoclonal anti 6-his antibody. Death is quantified by release of LDH, a cytoplasmic enzyme released in the culture medium when cells are dying. The read out is a colorimetric assay read at 490nm. T cells have been shown to be pathogenic in many autoimmune diseases and being able to control antigen- specific T cell death is a therapeutic strategy (e.g. anti-TNF ⁇ treatment in a patient with Crohn's disease).
  • Human-MLR proliferation and cytokine secretion:
  • This cell-based assay measures the effects of novel proteins on lymphocyte proliferation or inhibition of mouse spleen cells following stimulation by spleen cells from another donor (mouse strain). This cell-based assay measures the effect of novel proteins on T lymphocyte and antigen presenting cell responses and will be used to confirm activity of positives and hits identified in the human-MLR assays. This assay will be used to select proteins that will be tested in murine models of human diseases.
  • Human PBMC stimulated with the superantigen TSST
  • Superantigens are strong modulators of the immune system affecting T cells. Superantigens influence immunologically mediated disorders such as inflammatory bowel disease (IBD), inflammatory skin diseases like atopic dermatitis and psoriasis, hi this cellular assay, we are specifically targeting T lymphocyte activation via the TCR but with different requirements than the T cell response to classical antigens, in particular in respect to co-stimulatory molecules.
  • IBD inflammatory bowel disease
  • PHA Human PBMC stimulated with either ConA or PHA:
  • cytokine bead array CBA
  • IFN- ⁇ IFN- ⁇
  • TNF- ⁇ TNF- ⁇
  • IL-4 IL-4
  • IL-10 cytokine bead array
  • Most cytokines can have dual actions, pro or anti-inflammatory, depending of the injury, milieu and cellular target. Any protein with the capability to modulate cytokine secretion may have a therapeutic potential (e.g. decreasing IFN- ⁇ and TNF- ⁇ would be beneficial in ThI -mediated autoimmune disease. In contrast, decreasing IL-4, IL-5 may be beneficial in Th2- mediated-diseases. Inducing IL-10 would influential in MS and systemic lupus erythematosus (SLE)).
  • SLE systemic lupus erythematosus
  • This cell-based assay measures the effects of novel proteins on cytokine secretion (IFN- ⁇ , TNF- ⁇ ) induced by LPS acting on monocytes/macrophages and granulocytes. Any protein with the capability to modulate IFN- ⁇ and TNF- ⁇ secretion would be beneficial in ThI -mediated autoimmune diseases.
  • Neutrophils are important in inflammation and autoimmune diseases such as Rheumatoid Arthritis.
  • Leukocyte chemo-attractants such as IL-8 initiate a sequence of adhesive interactions between cells and the micro-vascular endothelium, resulting in activation, adhesion and finally migration of neutrophils.
  • the tissue infiltration of neutrophils depends on a reorganisation of cytoskeleton elements associated with specific changes in cell morphology of these cells. This cell-based assay measures the effect of novel proteins on cytoskeleton reorganization of human neutrophils.
  • B cell proliferation assay As well as infiltrating B cells, are thought to be important in the pathogenesis of various autoimmune diseases, such as systemic lupus erithematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome and myasthenia gravis. Compelling evidence indicates that a disregulation in B cell homeostasis could affect immune tolerance leading to the inappropriate survival of autoreactive B cells producing pathogenic antibodies and sustained inflammation. The identification of new factors that play critical roles in the regulation of B cell proliferation, survival and differentiation following B cell receptor triggering are of high relevance in the development of novel therapies. i) B cell proliferation assay:
  • This cell-based assay measures the effect of novel proteins on B cell co-stimulation.
  • the Ca + -flux in THPl -cell assay measures the effects of novel proteins on their ability to trigger an intracellular calcium release (a generic second messenger event) from the endoplasmic reticulum.
  • M-CSF is crucial for the final step of maturation of macrophages/microglia and is not replaceable by any other factor.
  • the evaluation of this biological response may represent a way to influence the microglial activity and therefore an opportunity to identify molecules with therapeutic potential fro MS.
  • a cell-based assay has been developed to measure the proliferative response of a microglia cell line to M-CSF. The feasibility and the robustness phases showed optimal results. This assay is in 96 well plates and is easily automated. Non-radioactive substrate is required.
  • MHC II in a microglia cell line as a response of IFN- ⁇ , TGF ⁇ and IL-10 can be evaluated for high throughput screening tests.
  • Any protein regulating MHC II expression may have an important role in controlling inflammation associated with CNS (the concept has been validated with statins).
  • the induction/inhibition of NO in microglia in a high throughput-screening test can be evaluated.
  • the modulation of the production of active molecules by microglia cells may represent an opportunity to diminish the cellular damage in the CNS.
  • First strand cDNA was prepared from human prostate total RNA sample (Clontech) using Thermoscript RT-PCR System (Invitrogen) according to the manufacturer's protocol.
  • Oligo (dT) 20 primer (l ⁇ l at 500 ⁇ g/ml) , 2.5 ⁇ g human total RNA, l ⁇ l 1OmM dNTP mix (1OmM each of dATP, dGTP, dCTP and dTTP at neutral pH) and sterile DEPC-treated water to a final volume of 12 ⁇ l were combined in a 0.2ml PCR tube, heated to 65°C for 5min and chilled on ice.
  • the contents were collected by brief centrifugation and 4 ⁇ l of 5X cDNA Synthesis Buffer, 2 ⁇ l 0.1 M DTT, l ⁇ l of sterile DEPC-treated water, l ⁇ l RnaseOUTTM Recombinant Ribonuclease Inhibitor (40units/ ⁇ l, Invitrogen) and l ⁇ l of ThermoScript RT enzyme (15units/ ⁇ l) were added.
  • the contents of the tube were mixed gently and incubated at 55 0 C for 1 hour and then inactivated by heating at 85°C for 5min.
  • l ⁇ l (2units) of E. coli RNase H (Invitrogen) was added and the reaction mixture incubated at 37°C for 30min.
  • PCR primer pairs having a length of between 18 and 30 bases were designed to amplify the predicted INSP170 cds. PCR primers were optimized to have a Tm close to 55 + 5°C and a GC content of 40-60%. Primers were selected which had high selectivity for the target sequence (INSP170/INSP222) with little or no non-specific priming.
  • Gene-specific cloning primers ESfSP 170fwd and ESfSP 170rev were designed to amplify cDNA fragments of 429 bp spanning the E ⁇ SP 170 cds and 264 bp spanning the ESfSP222 cds. (Table 4, Figure 6).
  • ESfSP 170 had been found to be over-expressed in prostate tissue
  • the primer pair was used to amplify products from prostate cDNA.
  • PCR was performed in a final volume of 25 ⁇ l containing IX Expand High FidelityTM buffer, 200 ⁇ M dNTPs, 50OnM of each cloning primer, lunit of Expand High Fidelity enzyme mix, and l ⁇ l of human prostate cDNA. Cycling was performed using an MJ Research DNA Engine, programmed as follows: 94°C, 2min; 35 cycles of 94°C, 45sec, 56°C, lmin, and 72 0 C, lmin 30sec; followed by 1 cycle at 72°C for lOmin and a holding cycle at 4°C.
  • PCR amplification reaction was visualised on a 2% agarose gel in 1 X TAE buffer (Promega). Products of approximately the expected molecular mass for ESfSP222 were identified. PCR reactions were then purified using the QIAquick Gel Extraction Kit (Qiagen), eluted in 30 ⁇ l of EB buffer (1OmM Tris) and exposed to a second round of PCR.
  • Product from the first round of PCR was then used as the template for the second round of PCR using the same amplification primers ES[SP 170fwd and ESfSP 170rev.
  • the second PCR reaction was performed in a final volume of 25 ⁇ l containing IX Expand High FidelityTM buffer, 200 ⁇ M dNTPs, 50pmoles of each cloning primer, 2.5unit of Expand High FidelityTM enzyme mix, (Roche), and 14 ⁇ l of gel purified PCR product from the first round of PCR.
  • PCR products were subcloned using TA cloning into the pGEM-T Easy cloning vector purchased from the Promega Corporation using a modification of the conditions specified by the manufacturer. Briefly, lO ⁇ l of gel purified PCR product was incubated over night at 4°C with 1 ⁇ l of pGEM-T Easy vector, 2 ⁇ l of T4 DNA ligase buffer, 6 ⁇ l of sterile water and l ⁇ l of T4 DNA ligase (New England Biolabs). The reaction mixture was then transformed into E.
  • coli strain DH5 ⁇ (Invitrogen) as follows: a 50 ⁇ l aliquot of DH5 ⁇ cells was thawed on ice and 5 ⁇ l of pGEM-T Easy reaction was added. The mixture was incubated for 1 hour on ice and then heat shocked by incubation at 42°C for exactly lmin. Samples were returned to ice and 500 ⁇ l of warm (room temperature) LB media was added. Samples were incubated with shaking (220 rpm) for Ih at 37°C. The transformation mixture was then plated on L-broth (LB) plates containing ampicillin (lOO ⁇ g/ml) and incubated overnight at 37°C.
  • LB L-broth
  • PCR Colonies were inoculated into 22.5 ⁇ l of PCR mix; 19.5 ⁇ l of sterile water, 2.5 ⁇ l of 10x Thermopol buffer (New England Biolabs) and 0.5 ⁇ l of 1OmM dNTPs.
  • PCR mix was subsequently heated to 99 0 C for 10 min and then allowed to cool to room temperature. This was made up to 25 ⁇ l volume containing lunit of T4 DNA Polymerase (New England Biolabs), 50OnM INSP170fwd and 50OnM INSP170rev, and subjected to PCR using an MJ Research DNA Engine.
  • the cycling conditions were as follows: 30 cycles of 94°C, 45sec, 52°C, lmin and 72°C for lmin. Samples were maintained at 4°C (holding cycle) before further analysis.
  • PCR reaction products were analyzed on 2% agarose gels in 1 X TAE buffer. Positive colonies, which gave PCR products of approximately the expected molecular weight, 264bp, were grown up overnight at 37°C in 3ml Terrific Broth containing ampicillin (lOO ⁇ g/ml), with shaking at 220 rpm. 3.7 Plasmid DNA preparation and sequencing
  • Miniprep plasmid DNA was prepared from 3ml cultures using QIAGEN Plasmid Mini Kit according to the manufacturer's instructions. Plasmid DNA was eluted in 50 ⁇ l of EB buffer (1OmM Tris). The DNA concentration was measured using a Beckman Coulter DU800 Spectrophotometer. Plasmid DNA (500ng) was sent to MRC-Geneservices and subjected to DNA sequencing with the sequencing primers M13F and M13R (Table 4).
  • Example 4 Exon structure of predicted and cloned sequences.
  • INSP223 is an amalgamation of the INSP 170 and INSP222 sequences and, although the Applicant does not wish to be bound by this theory, is predicted to be a full length, secreted variant of INSP 170, containing a shorter exon one, an alternative exon two, but maintaining both the Kazal domains and signal peptide of INSP 170.
  • Example 5 Expression and purification Further experiments may now be performed to determine the tissue distribution and expression levels of the INSP 170, INSP222 and INSP223 polypeptides in vivo, on the basis of the nucleotide and amino acid sequence disclosed herein.
  • the presence of the transcripts for INSP 170, INSP222 and INSP223 may be investigated by PCR of cDNA from different human tissues.
  • the INSP170, INSP222 and INSP223 transcripts may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result.
  • all RNA should be treated with DNAse prior to use for reverse transcription.
  • a control reaction may be set up in which reverse transcription was not undertaken (a -ve RT control).
  • RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers.
  • ABSI Multiscript reverse transcriptase
  • PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls.
  • INSP167-speciflc and INSP 170- specific primers may readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue.
  • any suitable cDNA libraries may be used to screen for the INSP 170, INSP222 and INSP223 transcripts, not only those generated as described above.
  • the tissue distribution pattern of the INSP 170, INSP222 and INSP223 polypeptides will provide further useful information in relation to the function of those polypeptides.
  • Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogen) are maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH). Sixteen to 20 hours prior to transfection
  • Scale-up batches may be produced by following the protocol called "PEI transfection of suspension cells", referenced BP/PEI/HH/02/04, with PolyEthylenelmine from Polysciences as transfection agent.
  • the culture medium sample containing the recombinant protein with a C-terminal 6His tag is diluted with cold buffer A (5OmM NaH 2 PO 4 ; 60OmM NaCl; 8.7% (w/v) glycerol, pH 7.5).
  • the sample is filtered then through a sterile filter (Millipore) and kept at 4 0 C in a sterile square media bottle (Nalgene).
  • the purification is performed at 4°C on the VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic).
  • the purification procedure is composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm, 0.83ml), followed by gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column (1,0 x 10cm).
  • the metal affinity column is regenerated with 30 column volumes of EDTA solution (10OmM EDTA; IM NaCl; pH 8.0), recharged with Ni ions through washing with 15 column volumes of a 10OmM NiSO 4 solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (5OmM
  • the Sephadex G-25 gel-filtration column is regenerated with 2ml of buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137mM NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20% (w/v) glycerol; pH 7.4).
  • buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; pH 7.2
  • buffer C 137mM NaCl; 2.7mM KCl; 1.5mM KH 2 PO 4 ; 8mM Na 2 HPO 4 ; 20% (w/v) glycerol; pH 7.4
  • the peak fraction eluted from the Ni-column is automatically loaded onto the Sephadex G-25 column through the integrated sample loader on the VISION and the protein is eluted with buffer C at a flow rate of 2 ml/min.
  • the fraction was filtered through a sterile centrifugation filter (Millipore), frozen and stored at -80°C.
  • An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) Western blot with anti- His antibodies.
  • the NuPAGE gel may be stained in a 0.1% Coomassie blue R250 staining solution (30% methanol, 10% acetic acid) at room temperature for Ih and subsequently destained in 20% methanol, 7.5% acetic acid until the background is clear and the protein bands clearly visible.
  • the proteins are electrotransferred from the gel to a nitrocellulose membrane.
  • the membrane is blocked with 5% milk powder in buffer E
  • the membrane is subsequently exposed to a Hyperfilm (Amersham Pharmacia), the film developed and the western blot image visually analysed.
  • the protein concentration may be determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.
  • overexpression or knock-down of the expression of the polypeptides in cell lines may be used to determine the effect on transcriptional activation of the host cell genome.
  • Dimerisation partners, co-activators and co-repressors of the ESfSPlVO, INSP222 and INSP223 polypeptides may be identified by immunoprecipitation combined with Western blotting and immunoprecipitation combined with mass spectroscopy.
  • Custom microarrays have been manufactured using Agilent Technologies' (Agilent Technologies Inc, Palo Alto, CA) non-contact in situ synthesis process of printing 60-mer length oligonucleotide probes, base-by-base, from digital sequence files. This is achieved with an inkjet process which delivers extremely small, accurate volumes (picoliters) of the chemicals to be spotted. Standard phosphoramidite chemistry used in the reactions allows for very high coupling efficiencies to be maintained at each step in the synthesis of the full-length oligonucleotide. Precise quantities are reproducibly deposited "on the fly.” This engineering feat is achieved without stopping to make contact with the slide surface and without introducing surface-contact feature anomalies, resulting in consistent spot uniformity and traceability. (Hughes et al. (2001) Nat. Biotech. Apr; 19(4): 342-7. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer).
  • cDNA synthesis and subsequent T7 polymerase amplification of Cyanine 3(5)-CTP labeled cRNA probe was carried out using Agilent's low RNA input fluorescent linear amplification kit from a template of 5 ⁇ g of total RNA according to the kit protocol (version 2 August 2003, Agilent, Palo Alto, CA). cRNA is then fragmented using Agilent's In Situ hybridization kit-plus and hybridized both according to Agilent's protocol (Agilent 60-mer oligo microarray processing protocol version 4.1 April 2004, Agilent, Palo Alto, CA). Microarray Chip Design
  • INSP 170 is a 5 exon prediction.

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Abstract

L'invention concerne de nouvelles protéines, désignées INSP 170, INSP222 et INSP223, identifiées comme de nouveaux membres humains de la famille Kazal des inhibiteurs de sérine protéase, et elle concerne en outre l'utilisation de ces nouvelles protéines et de séquences d'acides nucléiques provenant des gènes de codage pour le diagnostic, la prévention et le traitement de maladies.
PCT/GB2006/004846 2005-12-21 2006-12-21 Nouveaux membres de la famille kazal des inhibiteurs de serine protease WO2007072012A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009093119A2 (fr) * 2008-01-21 2009-07-30 Med Discovery S.A. Utilisation d'inhibiteurs de sérine protéases pour traiter des maladies de peau
EP2371857A1 (fr) * 2010-04-01 2011-10-05 CSL Behring GmbH Inhibiteurs de facteur XII pour traiter la maladie de poumon interstitiel
US8450275B2 (en) 2010-03-19 2013-05-28 Baxter International Inc. TFPI inhibitors and methods of use
US8466108B2 (en) 2008-12-19 2013-06-18 Baxter International Inc. TFPI inhibitors and methods of use
US8962563B2 (en) 2009-12-21 2015-02-24 Baxter International, Inc. TFPI inhibitors and methods of use
CN110585445A (zh) * 2019-10-17 2019-12-20 武汉工程大学 香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用
CN110753705A (zh) * 2017-06-30 2020-02-04 伊玛提克斯生物技术有限公司 新型t细胞受体及其免疫治疗
CN116042683A (zh) * 2023-01-10 2023-05-02 山西医科大学 一种重组旋毛虫丝氨酸蛋白酶抑制剂的制备方法及其应用

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Publication number Priority date Publication date Assignee Title
WO2003054152A2 (fr) * 2001-12-10 2003-07-03 Nuvelo, Inc. Nouveaux acides nucleiques et polypeptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003054152A2 (fr) * 2001-12-10 2003-07-03 Nuvelo, Inc. Nouveaux acides nucleiques et polypeptides

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009093119A2 (fr) * 2008-01-21 2009-07-30 Med Discovery S.A. Utilisation d'inhibiteurs de sérine protéases pour traiter des maladies de peau
WO2009093119A3 (fr) * 2008-01-21 2009-11-26 Med Discovery S.A. Utilisation d'inhibiteurs de sérine protéases pour traiter des maladies de peau
US11793864B2 (en) 2008-01-21 2023-10-24 Dermadis Sa Use of serine protease inhibitors in the treatment of skin diseases
AU2009207394B2 (en) * 2008-01-21 2015-01-22 Dermadis Sa Use of serine protease inhibitors in the treatment of skin diseases
US9873720B2 (en) 2008-12-19 2018-01-23 Baxalta GmbH TFPI inhibitors and methods of use
US9777051B2 (en) 2008-12-19 2017-10-03 Baxalta GmbH TFPI inhibitors and methods of use
US8466108B2 (en) 2008-12-19 2013-06-18 Baxter International Inc. TFPI inhibitors and methods of use
US11001613B2 (en) 2008-12-19 2021-05-11 Takeda Pharmaceutical Company Limited TFPI inhibitors and methods of use
US8962563B2 (en) 2009-12-21 2015-02-24 Baxter International, Inc. TFPI inhibitors and methods of use
US11793855B2 (en) 2010-03-19 2023-10-24 Takeda Pharmaceutical Company Limited TFPI inhibitors and methods of use
US9018167B2 (en) 2010-03-19 2015-04-28 Baxter International Inc. TFPI inhibitors and methods of use
US9556230B2 (en) 2010-03-19 2017-01-31 Baxalta GmbH TFPI inhibitors and methods of use
US8450275B2 (en) 2010-03-19 2013-05-28 Baxter International Inc. TFPI inhibitors and methods of use
US10201586B2 (en) 2010-03-19 2019-02-12 Baxalta GmbH TFPI inhibitors and methods of use
US9150638B2 (en) 2010-04-01 2015-10-06 Justus-Liebig-Universität Giessen Factor XII inhibitors for treating interstitial lung disease
JP2013527143A (ja) * 2010-04-01 2013-06-27 ユストゥス−リービッヒ−ウニヴェルジテート・ギーセン 間質性肺疾患を処置するための第ｘｉｉ因子阻害剤
WO2011121123A1 (fr) * 2010-04-01 2011-10-06 Csl Behring Gmbh Inhibiteurs de facteur xii pour traiter une maladie pulmonaire interstitielle
EP2371857A1 (fr) * 2010-04-01 2011-10-05 CSL Behring GmbH Inhibiteurs de facteur XII pour traiter la maladie de poumon interstitiel
US10800816B2 (en) 2012-03-21 2020-10-13 Baxalta GmbH TFPI inhibitors and methods of use
CN110753705A (zh) * 2017-06-30 2020-02-04 伊玛提克斯生物技术有限公司 新型t细胞受体及其免疫治疗
CN110585445A (zh) * 2019-10-17 2019-12-20 武汉工程大学 香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用
CN116042683A (zh) * 2023-01-10 2023-05-02 山西医科大学 一种重组旋毛虫丝氨酸蛋白酶抑制剂的制备方法及其应用
CN116042683B (zh) * 2023-01-10 2024-04-19 山西医科大学 一种重组旋毛虫丝氨酸蛋白酶抑制剂的制备方法及其应用

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