WO2007060431A1 - Proteine a domaine(s) ntr de type netrine - Google Patents

Proteine a domaine(s) ntr de type netrine Download PDF

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Publication number
WO2007060431A1
WO2007060431A1 PCT/GB2006/004376 GB2006004376W WO2007060431A1 WO 2007060431 A1 WO2007060431 A1 WO 2007060431A1 GB 2006004376 W GB2006004376 W GB 2006004376W WO 2007060431 A1 WO2007060431 A1 WO 2007060431A1
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seq
polypeptide
nucleic acid
disease
acid molecule
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PCT/GB2006/004376
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English (en)
Inventor
Kinsey Maundrell
Richard Mitter
David Michalovich
Richard Fagan
Christine Power
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Ares Trading S.A.
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Publication of WO2007060431A1 publication Critical patent/WO2007060431A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • NTR Netrin-like (NTR) Domain Containing Protein
  • This invention relates to novel proteins, termed INSP210 and INSP211 herein identified as Netrin-like (NTR) domain containing proteins, and to the use of these proteins and nucleic acid sequences from the encoding genes in the diagnosis, prevention and treatment of disease.
  • NTR Netrin-like domain containing proteins
  • Secreted Proteins The ability for cells to make and secrete extracellular proteins is central to many biological processes. Enzymes, growth factors, extracellular matrix proteins and signalling molecules are all secreted by cells. This is through fusion of a secretory vesicle with the plasma membrane. In most cases, but not all, proteins are directed to the endoplasmic reticulum and into secretory vesicles by a signal peptide. Signal peptides are cis-acting sequences that affect the transport of polypeptide chains from the cytoplasm to a membrane bound compartment such as a secretory vesicle. Polypeptides that are targeted to the secretory vesicles are either secreted into the extracellular matrix or are retained in the plasma membrane.
  • polypeptides that are retained in the plasma membrane will have one or more transmembrane domains.
  • secreted proteins that play a central role in the functioning of a cell are cytokines, hormones, extracellular matrix proteins (adhesion molecules), proteases, and growth and differentiation factors.
  • Netrin (NTR) like protein domains are characterized by 6 cysteines which form 3 disulphide bonds, and are present in a superfamily of extracellular, multi-domain proteins usually involved in proteolytically regulated pathways, such as netrins, complement proteins (C3, C4, C5), secreted frizzled related proteins (SFRP), WFIKKN multivalent protease inhibitors, procollagen C-proteinase enhancer proteins (PCOLCE).
  • Laminin domains, EGF-like domains, cysteine-rich frizzled domains, CUB domains, thrombospondin domains, ADAM spacer and ADAM cysteine rich domains, follistatin, SEA, immunoglobulin, lcunitz, whey acidic protein (WAP), kazal and A-macroglobulin domains are amongst the other domains included in these proteins, and some of them are known to inhibit proteases (Liepinsh E et al, 2003, J. Biol. Chem. 278:25982-25989).
  • the invention is based on the discovery that the INSP210 and INSP211 polypeptides are NTR domain containing proteins.
  • the invention describes a number of sequences and partial sequences of these polypeptides. These are listed in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
  • the INSP210 polypeptide has three splice variants, referred to herein as “the INSP211 polypeptide", “the INSP210SV1 polypeptide” and “the INSP210SV2 polypeptide”. Whilst the first 2 exons of the INSP211 polypeptide are the same as the first 2 exons of the INSP210 polypeptide, INSP211 has an extended coding exon 3, which introduces an early stop codon and thus truncates the encoded protein.
  • the constituent amino acid sequences of this splice variant are recited in SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12.
  • the INSP210SV1 polypeptide is identical to INSP210 except that it lacks the sequence encoded by exon 2. This deletion removes 3 of the 6 conserved cysteines found in the netrin domain.
  • the constituent amino acid sequences of this splice variant are recited in SEQ ID NO:14 and SEQ ID NO:18.
  • the INSP210SV2 polypeptide is identical to INSP210 except that it lacks the sequence encoded by exons 2 and 3.
  • the constituent amino acid sequences of this splice variant are recited in SEQ ID NO: 16 and SEQ ID NO:20.
  • polypeptide which polypeptide: (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 ED NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ IDNO:20;
  • (ii) is a fragment thereof which is a member of the NTR domain containing protein family, or has an antigenic determinant in common with the polypeptides of (i); or (iii) is a functional equivalent of (i) or (ii).
  • the polypeptide according to this first aspect of the invention comprises the amino acid sequence as recited in SEQ ID NO:2 or SEQ ID NO:8.
  • polypeptide which consists of the amino acid sequence as recited in SEQ ED NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ED NO: 10, SEQ ID NO: 12, SEQ ED NO: 14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20.
  • the polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as the "INSP210 full protein sequence".
  • the polypeptide having the sequence recited in SEQ ID NO:4 is referred to hereafter as the "mature INSP210 protein sequence".
  • the polypeptide having the sequence recited in SEQ ID NO:6 is referred to hereafter as the "INSP210 NTR domain protein sequence”.
  • the polypeptide having the sequence recited in SEQ ID NO: 8 is referred to hereafter as the "INSP211 full protein sequence”.
  • the polypeptide having the sequence recited in SEQ ID NO: 10 is referred to hereafter as the "mature INSP211 protein sequence”.
  • the polypeptide having the sequence recited in SEQ ID NO: 12 is referred to hereafter as the "INSP211 NTR domain protein sequence".
  • the polypeptide having the sequence recited in SEQ ID NO:14 is referred to hereafter as the "INSP210SV1 full protein sequence”.
  • the polypeptide having the sequence recited in SEQ ID NO: 16 is referred to hereafter as the "INSP210SV2 full protein sequence”.
  • the Applicant does not wish to be bound by this theory, it is postulated that the first 17 amino acids of the INSP210 and INSP211 polypeptides form a signal peptide.
  • the full length INSP210 polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO:4.
  • the full length INSP211 polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO:10.
  • the full length INSP210SV1 polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO: 18.
  • the full length INSP210SV2 polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO:20.
  • the term "INSP210 polypeptides” as used herein includes polypeptides comprising the INSP210 full protein sequence, the mature INSP210 protein sequence, the INSP210 NTR domain protein sequence, the full length INSP210SV1 protein sequence, the mature INSP210SV1 protein sequence, full length INSP210SV2 protein sequence and the mature INSP210SV2 protein sequence.
  • the term "INSP211 polypeptides” as used herein includes polypeptides comprising the INSP211 full protein sequence, the mature INSP211 protein sequence and the INSP211 NTR domain protein sequence.
  • Netrin-like (NTR) domain containing protein refers to a molecule containing at least one Netrin-like (NTR) domain.
  • the "Netrin-like (NTR) domain containing protein” may be a molecule containing a Netrin-like (NTR) domain detected with an e- value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
  • NTR domain containing protein may be a molecule matching the HMM build of the Pfam entry detected with an e-value lower than 0.1, 0.01, 0.00I 5 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.
  • a polypeptide according to the above embodiments of the first aspect of the invention functions as a secreted protein, particularly as a member of the NTR domain containing protein family.
  • a Netrin-like (NTR) domain containing protein shows biological activity in at least one of the assays described in US6670451 and US6087326.
  • Activity of the polypeptides of the invention e.g. INSP210 or INSP211, can be evaluated by measuring activity in any one of these assays.
  • the polypeptides of the first aspect of the invention may further comprise a histidine tag.
  • the histidine tag is found at the C-terminal of the polypeptide.
  • the histidine tag comprises 1-10 histidine residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably the histidine tag comprises 6 histidine residues.
  • An "antigenic determinant" of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR).
  • 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 INSP210, INSP211 or a fragment thereof.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the "antigenic determinant" refers to a particular chemical group on a polypeptide of the present invention that is antigenic, i.e. that elicit a specific immune response.
  • AAF44924 a breast cancer related sequence (matches amino acids 64 -276; herein referred to as SEQ ID NO:38), ADM67135, a human homologue of murine adipocyte specific cDNA (amino acids 159-339) and ABP60093, a novel protease inhibitor (match over amino acids 243-339).
  • the invention provides a purified nucleic acid molecule which encodes a polypeptide according to the first aspect of the invention.
  • purified nucleic acid molecule preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
  • genomic DNA are specifically excluded from the scope of the invention.
  • genomic DNA larger than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp are specifically excluded from the scope of the invention.
  • the "purified nucleic acid molecule" consists of cDNA only.
  • the purified nucleic acid molecule comprises the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP210 full protein sequence), SEQ ID NO:3 (encoding the mature INSP210 protein sequence), SEQ ID NO:5 (encoding INSP210 NTR domain protein sequence), SEQ ID NO:7 (encoding the INSP211 full protein sequence), SEQ ID NO:9 (encoding the mature INSP211 protein sequence), SEQ ID NO:11 (encoding the INSP211 NTR domain protein sequence), SEQ ID NO: 13 (encoding the full length INSP210SV1 protein sequence), SEQ ID NO:17 (encoding the mature INSP210SV1 protein sequence), SEQ ID NO:15 (encoding the full length INSP210SV2 protein sequence) or SEQ ID NO: 19 (encoding the mature INSP210SV2 protein sequence), or is a redundant equivalent or fragment of any one of these sequences.
  • the invention further provides that the purified nucleic acid molecule consists of the nucleic acid sequences as recited in SEQ ID NO:1 (encoding the INSP210 full protein sequence), SEQ ID NO:3 (encoding the mature INSP210 protein sequence), SEQ ID NO:5 (encoding INSP210 NTR domain protein sequence), SEQ ID NO:7 (encoding the INSP211 full protein sequence), SEQ ID NO:9 (encoding the mature INSP211 protein sequence), SEQ ID NO:11 (encoding the INSP211 NTR domain protein sequence) ), SEQ ID NO: 13 (encoding the full length INSP210SV1 protein sequence), SEQ ID NO: 15 (encoding the mature INSP210SV1 protein sequence), SEQ ID NO: 17 (encoding the full length INSP210SV2 protein sequence) or SEQ ID NO: 19 (encoding the mature INSP210SV2 protein sequence), or is a redundant equivalent or fragment of any one of these sequences.
  • AK026583 from signet ring cell carcinoma herein referred to as SEQ ID NO: 39
  • BC036760 from ovary herein referred to as SEQ ID NO;40
  • the invention provides a purified nucleic acid molecule which hybridises under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the invention provides a ligand which binds specifically to members of the NTR domain containing protein family 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 NTR domain containing protein family.
  • 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 INSP210 and INSP211 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease.
  • Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.
  • Another aspect of this invention resides in the use of an INSP210 or INSP211 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against Netrin-like (NTR) domain containing protein related disorders.
  • NTR Netrin-like
  • a further aspect of this invention resides in methods of screening of compounds for therapy of Netrin-like (NTR) domain containing protein related disorders, comprising determining the ability of a compound to bind to an INSP210 or INSP211 gene or polypeptide, or a fragment thereof.
  • NTR Netrin-like
  • a further aspect of this invention resides in methods of screening of compounds for therapy of Netrin-like (NTR) domain containing protein related disorders, comprising testing for modulation of the activity of an INSP210 or INSP211 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 NTR domain containing protein family are implicated.
  • Such diseases may include cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours (see Baker, A.H. et al., J. Cell.
  • 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, asthma, 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, Alzheimer's 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.
  • myeloproliferative disorders such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angio
  • the diseases are those in which NTR domain containing proteins are implicated. These molecules may also be used in the manufacture of a medicament for the treatment of such diseases. These molecules may also be used in contraception or for the treatment of reproductive disorders including infertility.
  • the moieties of the present invention i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • 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 invention provides for the use of a polypeptide of the first aspect of the invention as an NTR domain containing protein.
  • Suitable uses of the polypeptides of the invention as NTR domain containing proteins include use as a regulator of cellular growth, metabolism or differentiation, use as part of a receptor/ligand pair and use as a diagnostic marker for a physiological or pathological condition.
  • 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, proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours (see Baker, A.H. et al., J. Cell.
  • 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, asthma, 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, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellirus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions.
  • the diseases are those in which NTR domain containing proteins are implicated.
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist.
  • antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
  • the INSP210 and INSP211 polypeptides are Netrin-like (NTR) domain containing proteins and thus have roles in many disease states. Antagonists of the INSP210 and INSP211 polypeptides are of particular interest as they provide a way of modulating these disease states.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be used in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • “functional equivalent” refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention.
  • a functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional equivalent” is intended to include the fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.
  • the "functional equivalent” may be a protein or nucleic acid molecule that exhibits any one or more of the functional activities of the polypeptides of the present invention.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays substantially similar activity compared with INSP210, INSP211 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 INSP210, INSP211 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 INSP210, INSP211 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%.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • a fusion protein may contain one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • the polypeptide of the invention comprising a sequence having at least 85% of homology with INSP210 or INSP211 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 INSP210 or INSP211 in frame to the coding sequences for a heterologous protein sequence.
  • Preferred polypeptides that comprise a sequence having at least 85% homology with INSP210 or INSP211 are specific regions of the polypeptides such as the INSP210 and INSP211 NTR domain protein sequences.
  • heterologous when used herein, is intended to designate any polypeptide other than a human INSP210 or INSP211 polypeptide.
  • Example of heterologous sequences, that can be comprised in the soluble fusion proteins either at N- or at C-terminus, are the following: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc region), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences allowing purification by affinity chromatography.
  • heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in the fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003).
  • 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., Proc Natl Acad Sci USA, 86: 821-4,
  • the heterologous sequence can be eliinated 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.
  • 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.
  • 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:41) introduced between the sequence of the substances of the invention and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • the protein is fused to the constant region of an Ig molecule.
  • it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgGl, for example.
  • Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms 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.
  • modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods (see, for example, Bray 2003, Nat Rev Drug Discov, 2(7):587-93; Casi & Hilvert 2003, Curr Opin Struct Biol, 13(5):589-94).
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the INSP210 and INSP211 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 INSP210 and INSP211 polypeptides.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Such substitutions are among Ala, VaI, Leu and lie; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • any substitution should be preferably a "conservative” or “safe” substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties (e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.
  • non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the Netrin-like (NTR) domain containing protein may increase its ability to be reused and recycled, potentially increasing its therapeutic potency (Robinson CR, 2002). Immunogenic epitopes eventually present in the polypeptides of the invention can be exploited for developing vaccines
  • Step S, 2002 or eliminated by modifying their sequence following known methods for selecting mutations for increasing protein stability, and correcting them (van den Burg B and Eijsink V, 2002; WO 02/05146, WO 00/34317, WO 98/52976).
  • amino acids derivatives included in peptide mimetics are those defined in Table 2.
  • a non-exhaustive list of amino acid derivatives also include aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydro- isoquinoline-3-COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidine-4- carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine, and phenylglycine.
  • amino acid derivative is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids.
  • the amino acid derivative may contain substituted or non-substituted, linear, branched, or cyclic alkyl moieties, and may include one or more heteroatoms.
  • the amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem- Novabiochem AG, Switzerland; Bachem, USA).
  • polypeptides of the first aspect of the invention have a degree of sequence identity with the INSP210 and INSP211 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 (see PCT application WO 01/69507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP210 and INSP211 polypeptides, are predicted to be members of the NTR domain containing protein family, by virtue of sharing significant structural homology with the INSP210 and INSP211 polypeptide sequence.
  • significant structural homology is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.
  • the polypeptide of the first aspect of the invention also include fragments of the INSP210 and INSP211 polypeptides and fragments of the functional equivalents of the INSP210 and INSP211 polypeptides, provided that those fragments are members of the NTR domain containing protein family or have an antigenic determinant in common with the INSP210 and INSP211 polypeptide.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP210 and INSP211 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 1-350 amino acids in length, preferably 3-200, 5-100, 7-50, or 9-20 amino acids.
  • Nucleic acids according to the invention are preferably 10-2100 nucleotides in length, preferably 20-1500 nucleotides, preferably 50-1000, 100-600, 200-550, 300-500 nucleotides in length.
  • Polypeptides according to the invention are preferably 5-500 amino acids in length, 50-400, 100-300, or 150-250 amino acids in length.
  • Fragments of the full length INSP210 and INSP211 polypeptides may consist of combinations of 2, 3, or all 4 neighbouring exon sequences in the INSP210 polypeptide sequence, or 2, or all 3 neighbouring exon sequences in the INSP211 polypeptide sequence. These exons may be combined with further mature fragments according to the invention. For example, such combinations include exons 1 and 2, exons land 3, exons 1, 2 and 4 and so on. Such fragments are included in the present invention. Fragments may also consist of combinations of different domains of the INSP210 and INSP211 proteins.
  • Preferred fragments of the INSP210 polypeptide sequence include the extracellular and intracellular forms discussed above. Further preferred fragments include those including or consisting of one or more NTR domains. The boundary of this domain is illustrated in Figure 1.
  • 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 secreted proteins.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, 10 4 - fold, 10 5 -fold, 10 6 -fold or greater for a polypeptide of the invention than for known secreted proteins such as members of the NTR domain containing protein family.
  • NTR Netrin-like
  • 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.
  • 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 at, 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 at, 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 at, (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.
  • 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 and SEQ ID NO:20, and functionally equivalent polypeptides.
  • These 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.
  • nucleic acids that encode entire INSP210 or 211 polypeptides (or domains thereof) by exon assembly.
  • each of the DNA segments corresponding to the complete or partial exon(s) that make up the gene sequence may be amplified separately.
  • INSP211 might be generated from human genomic DNA using PCR primers designed on the basis of the sequences of the constituent 3 coding exons, the first two corresponding to exons 1 and 2 of INSP210 transcript, and third one resulting from the 3' extension of exon 3 in INSP210 transcript. These fragments can be then assembled in an appropriately ordered manner by further PCR and restriction site manipulations into an appropriate expression vector, optionally including, for example, an heterologous signal peptide situated 5' to these fragments.
  • the nucleic acid molecules may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • nucleic acid molecule also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drag 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 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 and SEQ ID NO:20 5 .
  • 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. ScL, 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.
  • 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 INSP210 and INSP211 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 INSP210 and INSP211 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 hybridising 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 hybridisation probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INSP210 and INSP211 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the INSP210 and INSP211 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 JD NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19), 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.
  • probes the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al, PNAS USA 85, 8998-9002, 1988).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size- selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation, hi this technique, a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation. Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • RNA interference RNA interference
  • Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.
  • the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al. ⁇ supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et ah, ⁇ supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • HACs Human artificial chromosomes
  • the vectors pCR4-TOPO, pCR4-TOPO-INSP210, pENTR, pENTR_INSP210EC-6HIS, pEAK12d-PAC, pDEST12.2, pEAK12d-PACJNSP210EC-6HIS and pDEST12.2_INSP210EC-6HIS are preferred examples of suitable vectors for use in accordance with the aspects of this invention relating to INSP210.
  • 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.
  • control sequences it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells may be cloned into the vector.
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • the control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C 127, 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 aprf ⁇ cells, respectively. Also, 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.
  • DHFR dihydrofolate reductase
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. MoI. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al.
  • 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.
  • a vector for the production of an mRNA probe Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate 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 which may be used for ease of detection, include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif.
  • the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. 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 INSP210 or INSP211 polypeptide as a target for screening candidate drugs for treating or preventing Netrin-like (NTR) domain containing protein related disorders.
  • Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with an INSP210 or INSP211 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.
  • a further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a INSP210 or INSP211 polypeptide with a candidate compound, and selecting compounds that bind said INSP210 or INSP211 polypeptide at the surface of said cells and/or that modulate the activity of the INSP210 or INSP211 polypeptide.
  • a “biologically active” compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound having Netrin-like
  • NTR Netrin-like domain containing protein activity
  • a "biologically active” compound preferably is a compound that modulates the activity of INSP210 or INSP211.
  • 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 Netrin-like (NTR) domain containing protein related disorder, such as an animal model.
  • Preferred selected compounds are agonists of INSP210 or INSP211, i.e., compounds that can bind to INSP210 or INSP211 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 INSP210 or INSP211 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INSP210 or INSP211 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 an INSP210 or INSP211 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INSP210 or INSP211 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 INSP210 or INSP211 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 ah, 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 Netrin-like (NTR) domain containing protein related disorder in a subject.
  • the determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc.
  • the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.).
  • Modulation of activity includes, without limitation, stimulation of the surface expression of the INSP210 or INSP211 receptor, modulation of multimerization of said receptor (e.g., the formation of multimeric complexes with other sub-units), etc.
  • the cells used in the assays may be any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a INSP210 or INSP211 polypeptide) or any cell that expresses an endogenous INSP210 or INSP211 polypeptide. Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • E.coli E.coli, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
  • mammalian cell lines e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a particular example is cotransfecting a construct expressing a polypeptide according to the invention, or a fragment such as the LBD, in fusion with the GAL4 DNA binding domain, into a cell together with a reporter plasmid, an example of which is pFR-Luc (Stratagene Europe, Amsterdam, The Netherlands).
  • This particular plasmid contains a synthetic promoter with five tandem repeats of GAL4 binding sites that control the expression of the luciferase gene. When a potential ligand is added to the cells, it will bind the GAL4-polypeptide fusion and induce transcription of the luciferase gene.
  • the level of the luciferase expression can be monitored by its activity using a luminescence reader (see, for example, Lehman et al. JBC 270, 12953, 1995; Pawar et al. JBC, 277, 39243, 2002).
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises: (a) contacting a labelled or unlabeled compound with the polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself; or
  • a method such as FRET detection of ligand bound to the polypeptide in the presence of peptide co-activators might be used.
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises: (a) contacting a cell expressing on the surface thereof the polypeptide, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and
  • 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: (a) incubating a labelled ligand with a whole cell expressing a polypeptide according to the invention on the cell surface, or a cell membrane containing a polypeptide of the invention,
  • step (c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium; (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c); and
  • step (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • a method of screening for a polypeptide antagonist or agonist compound which 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;
  • step (d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c); and (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.
  • the INSP210 and INSP211 polypeptides of the present invention may modulate cellular growth and differentiation.
  • the biological activity of the INSP210 and INSP211 polypeptides can be examined in systems that allow the study of cellular growth and differentiation such as organ culture assays or in colony assay systems in agarose 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
  • the effect of the INSP210 and INSP211 polypeptides upon cellular differentiation can be measured by contacting stem cells or embryonic cells with various amounts of the INSP210 and INSP211 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 INSP210 and INSP211 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 INSP210 and INSP211 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 INSP210 and INSP211 polypeptides, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the INSP210 and INSP211 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.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays.
  • Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signaling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • 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 INSP210 or INSP211 polypeptide or fragment thereof, whereby the fragment is preferably a INSP210 or INSP211 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP210 or INSP211 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; 2. a bispecific or multispecif ⁇ c antibody,
  • an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).
  • a fibronectin-based binding molecule e.g. trinectin or adnectin.
  • Anticalins are also known in the art (Vogt et al., 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224. Furthermore, the test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
  • an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
  • the various moieties of the invention i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention
  • the various moieties of the invention may be useful in the therapy or diagnosis of diseases.
  • one or more of the following assays may be carried out.
  • test compound refers to the test compound as being a protein/polypeptide
  • test compound a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound”.
  • compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
  • the pharmaceutical compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a 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.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions of 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. Once formulated, the compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • an inhibitor compound as described above
  • a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • antagonists are antibodies.
  • such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question may be administered.
  • the polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • triplex DNA Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) hi: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY).
  • the complementary sequence or antisense molecule may also be designed to block translation of niRNA by preventing the transcript from binding to ribosomes.
  • Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules.
  • ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
  • in vzvo 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.
  • nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included.
  • Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • 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 radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations 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 ah, Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
  • mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane.
  • 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 al).
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays).
  • This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex. Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation
  • the amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules known in the art may be used, several of which are described above.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise:
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease in which members of the NTR domain containing protein molecule family are implicated.
  • diseases may include cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours (see Baker, A.H. et al., J. Cell.
  • 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, asthma, 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, Alzheimer's 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.
  • the diseases are those in which lymphocyte antigens are implicated.
  • lymphocyte antigens are implicated.
  • Figure 2 Alignment of INSP210 and INSP211 protein sequences with the most similar sequence; the predicted signal peptide in INSP210 and INSP211 is underlined; the predicted NTR domains are boxed.
  • Figure 3 Alignment of the NTR domains of INSP210 (residues 193-338) and INSP211 (residues 193-294) with other human NTR domain containing proteins: procollagen C- endopeptidase enhancer 1 (PCOLCE l-NP_002584, residues 327-437), procollagen C- endopeptidase enhancer 2 (PCOLCE2-NP_037495, residues 297-415), WFIKKNl ( WAP, FS, Ig 5 KU, and NTR-containing protein; WFKKN-NP_444514, residues 377-540), Netrin 1 (NETRINl-NP_004813, residues 472-601). The six characteristic cysteines are evidenced.
  • Figure 4 INSP210 cDNA and protein sequence. The position of PCR primers used for cloning is indicated by arrows.
  • Figure 5 Nucleotide sequence with translation of the INSP210SV1 PCR product.
  • Figure 6 Nucleotide sequence with translation of the INSP210SV2 PCR product.
  • Figure 7 Clustal W nucleotide sequence alignment of the predicted INSP210 sequence with the cloned splice variants INSP210SV1 and INSP210SV2.
  • Figure 8 Clustal W amino acid sequence alignment of the predicted INSP210 sequence with the cloned splice variants INSP210SV1 and INSP210SV2.
  • Example 1 Identification and in silico analysis of INSP210 and INSP211
  • the INSP210 (SEQ ID NO:2) and INSP211 (SEQ ID NO:8) are protein sequences of 339 and 317 amino acids, respectively, resulting from the alternative assembly of exonic sequences in the human genome.
  • INSP210 results from the transcription of 4 coding exons.
  • the INSP211 variant results from the transcription of 3 coding exons, the first two corresponding to exons 1 and 2 of INSP210 transcript, and third one resulting from the 3' extension of exon 3 in INSP210 transcript.
  • procollagen C-endopeptidase enhancer procollagen, type 1 and 2 are extracellular glycoproteins that enhances the activities of procollagen C-proteinases such as bone morphogenic protein 1 (BMPl).
  • BMPl bone morphogenic protein 1
  • This function is probably mediated by the CUB domains binding to the C-terminal propeptide of typel procollagen as the enhancer activity occurs even if the NTR domain is removed (Takahara K et al., 1994 J. Biol. Chem. 269: 26280-26285; Mott JD et al., 2000, J Biol Chem., 275:1384-90; Ricard-Blum S et al., 2002, J. Biol. Chem. 277: 33864-33869; Steiglitz B et al., 2002, J. Biol. Chem. 277:49820-
  • WAP WFIKKNl
  • FS FS, Ig, KU, and NTR-containing protein
  • NTR-containing protein is a secreted multidomain protein including, a WAP domain, a follistatin domain, an immunoglobulin domain, two tandem Kunitz domains, and an NTR domain. These domains have been implicated frequently in inhibition of various types of proteases, suggesting that the encoded protein may be a multivalent protease inhibitor and may control the action of multiple types of serine proteases as well as metalloproteinases (Nagy A et al., 2003, Eur J Biochem, 270: 2101-7; Trexler M et al, 2001, Proc Natl Acad Sci U S A, 98:3705-9).
  • Netrin 1 is thought to be involved in axon guidance and cell migration during development. Mutations and loss of expression of netrin suggest that variation in netrin may be involved in cancer development (Serafini, T et al., 1996, Cell 87 (6),
  • INSP210 and INSP211 cDNA and/or gene from samples of tissues of human origin will allow their high level expression in prokaryotic or eukaryotic expression systems and their subsequent purification and characterization as recombinant proteins.
  • Alternative recombinant INSP210 and INSP211 sequences can be produced by cloning in the appropriate expression vector the DNA encoding for full length INSP210 (SEQ ID NO: 2) or INSP211 (SEQ ID NO: 8), or for selected portions such as their respective mature sequences (SEQ ID NO: 4 and SEQ ID NO: 10), or their respective isolated NTR-like domains (SEQ ID NO: 6 and SEQ ID NO: 12).
  • heterologous DNA sequences encoding for protein sequences helping the expression, the secretion, and/or the detection of these recombinant sequences may be added in frame to the DNA encoding the INSP210- and INSP211 -derived proteins.
  • heterologous sequences are signal peptides and histidine tags.
  • Recombinant INSP210- and INSP211 -derived proteins may be used to generate INSP210- and INSP211 -specific monoclonal or polyclonal antibodies that might then be used in the biochemical characterisation of INSP210 and INSP211.
  • recombinant INSP210 and INSP211 may be used in a wide variety of screening assays.
  • Example 2 Cloning of INSP210SV1
  • PCR primers were designed for amplifying the predicted coding sequence of the virtual cDNA using Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). Primers were selected which had high selectivity for the target sequence (INSP210).
  • First strand cDNA was prepared from a variety of human tissue total RNA samples (Clontech, Stratagene, Ambion, Biochain Institute and in-house preparations) using Superscript II or Superscript III RNase H " Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol.
  • a cDNA synthesis mix was prepared as follows: 2 ⁇ l 1OX RT buffer, 4 ⁇ l 25mM MgCl 2 , 2 ⁇ l 0.1 M DTT, 1 ⁇ l RNaseOUTTM (40 U/ ⁇ l) and 1 ⁇ l Superscript IIITM RT enzyme were combined in a separate tube and then 10 ⁇ l of this mix added to the tube containing the RNA/primer mixture. The contents of the tube were mixed gently, collected by brief centrifugation, and incubated at 50 0 C for 50 min. The reaction was terminated by incubating at 80 °C for 5 min and the reaction mixture then chilled on ice and collected by brief centrifugation. To remove RNA complementary to the cDNA, l ⁇ l (2 units) of E. coli RNase H (Invitrogen) was added and the reaction mixture incubated at 37 °C for 20 min.
  • the final 21 ⁇ l reaction mix was diluted by adding 179 ⁇ l sterile water to give a total volume of 200 ⁇ l. This represented approximately 20 ng/ ⁇ l of each individual cDNA template.
  • Gene-specific cloning primers (INSP210-F1 and INSP210-R1, Table 3, and Figure 4) were designed to amplify a cDNA fragment of 1080 bp containing the predicted ORF of INSP210.
  • the primer pair was used to screen human cDNA templates and libraries derived from colon, ovary and pancreas as well as the universal reference RNA.
  • PCR reactions were performed in a final volume of 50 ⁇ l containing IX Taq High fidelity buffer, 200 ⁇ M dNTPs, 2 mM MgSO 4 , 0.2 ⁇ M of each cloning primer, 5 ⁇ l of 1OX enhancer solution, 0.2 ⁇ l (1 unit) of Taq High fidelity polymerase, and 1 ⁇ l of each cDNA template. Cycling was performed using an MJ Research DNA Engine, programmed as follows: 94 °C, 2 min; 40 cycles of 94 °C, 30 sec, 60 °C, 30 sec, and 68°C, 2 min; followed by 1 cycle at 68 0 C for 8 min and a holding cycle at 4 °C.
  • PCR reaction was then performed on 1 ul of PCR products from PCR 1 under the same reaction conditions as for PCRl, except that nested PCR primers (INSP210-F1 nest and INSP210-R1 nest) were used.
  • PCR products 40 ⁇ l were visualized on a 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) and purified using the MinElute Gel extraction kit (Qiagen), eluted in 10 ⁇ l of nuclease free water and subcloned directly.
  • PCR products were subcloned into the topoisomerase I modified cloning vector (pCR4-TOPO) using the TA cloning kit purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 ⁇ l of gel purified PCR product was incubated for 15 min at room temperature with 1 ⁇ l of TOPO vector and 1 ⁇ l salt solution. The reaction mixture was then transformed into E. coli strain TOPlO (Invitrogen) as follows: a 50 ⁇ l aliquot of One Shot TOPlO cells was thawed on ice and 2 ⁇ l of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 °C for exactly 30 s.
  • TOPO E. coli strain TOPlO
  • Samples were returned to ice and 250 ⁇ l of warm (room temperature) SOC media was added. Samples were incubated with shaking (220 rpm) for 1 h at 37 °C. The transformation mixture was then plated on L-broth (LB) plates containing ampicillin (100 ⁇ g/ml) and incubated overnight at 37 °C.
  • LB L-broth
  • PCR products were analyzed on 1 % agarose gels in 1 X TAE buffer. Colonies which gave PCR products of approximately the expected molecular weight were grown up overnight at 37 °C in 5 ml L-Broth (LB) containing ampicillin (100 ⁇ g /ml), with shaking at 220 rpm. 2.6 Plasmid DNA preparation and sequencing
  • Colonies which gave the expected band size by colony PCR were inoculated into 5 ml L- Broth (LB) containing ampicillin (100 ⁇ g /ml) and grown overnight at 37 °C with shaking at 220 rpm.
  • Miniprep plasmid DNA was prepared from the 5 ml culture using a Biorobot 8000 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l of sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices).
  • Plasmid DNA 200-500 ng was subjected to DNA sequencing with the T7 and T3 primers using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions.
  • the primer sequences are shown in Table 3. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • PCR products corresponding to the predicted INSP210 sequence were identified in 4 clones analysed from Sl 17 (cDNA derived from the universal reference RNA sample). The sequences were identical to the original prediction except that exon 2 was deleted.
  • INSP210SV a splice variant of INSP210, named INSP210SV1.
  • the sequence of the cloned INSP210SV cDNA fragment is shown in Figure 5.
  • a plasmid containing the sequence verified PCR product is pCR4-TOPO-INSP21 OSVl -Sl 17-3.
  • RNA was prepared by mixing approximately 10 ⁇ g total RNA from each of the following sources: Brain (Clontech), Heart (Clontech), Kidney (Clontech), Liver (Clontech), Lung (Clontech), Placenta (Clontech), Skeletal Muscle (Clontech), Small Intestine (Clontech), Spleen (Clontech), Thymus (Clontech), Uterus (Clontech) Bone Marrow (Clontech) Thyroid (Clontech), Ovary (Ambion), Prostate (Ambion), Skin (Resgen), Pancreas (Clontech), Salivary gland (BD Biosciences), Adrenal gland (BD Biosciences), Breast (Ambion), Pituitary gland (BioChain Institut), Stomach (Ambion), Mammary gland (Clontech), Lymph Node (BioChain Institut),
  • RNA was fractionated by chromatography on a pre-packed oligo-dT column (Stratagene) according to the protocol supplied by the manufacturer. Approximately 400 ⁇ g total RNA yielded 12.6 ⁇ g polyA+ mRNA which was aliquoted and stored frozen at -8O 0 C.
  • the gene specific cDNA primer for INSP210, AS403, was pooled with gene specific cDNA primers for 9 other predictions, each at a final concentration of 1 pM.
  • the pooled cDNA primer set was diluted 10 fold into 50 ⁇ l of a mixutre containing 1 x RT buffer, 500 ⁇ M each dNTPs, lOU/ ⁇ l RNAguard (Pharmacia) and 1 ⁇ g denatured polyA + RNA (prepared as described above).
  • cDNA synthesis was initiated by addition of 1OU Omniscript reverse transcriptase (Qiagen) and allowed to proceed for 1 h at 37 0 C. At the end of the reaction, 5 ⁇ l of the cDNA mix was used for PCR amplification as described below.
  • PCR primers were designed to span the entire predicted coding sequence of INSP210. BamHl restriction sites were added at the 5' end of each primer since no internal sites for this enzyme were predicted. A reaction mixture was set up containing 1 x PCR buffer, 0.2 mM each dNTP, 0.5 ⁇ M each PCR primer, 5 ⁇ l cDNA template, and the PCR reaction was initiated by addition of 5U PfuTurbo (Stratagene).
  • Cycling conditions were: 95°C, 3min (1 cycle); 95°C, 30 sec; 5O 0 C, 30 sec and 75 0 C, 70 sec (35 cycles); 75°C, 10 min (1 cycle).
  • An aliquot of the PCR reaction was analysed by electrophoresis on 0.8% agarose gels and the remainder was purified using the Wizard PCR Cleanup System (Promega) as recommended by the manufacturer, prior to subcloning of the PCR products.
  • the precipitated DNA was recovered by centrifugation, washed in 70% ethanol, dried under vacuum and ligated in a final volume of 10 ⁇ l using the Rapid Ligation Kit (Roche Diagnostics) according to the protocol supplied by the manufacturer.
  • the ligation mixture was then used to transform E. coli strain JMlOl as follows: 50 ⁇ l aliquots of competent JMlOl cells were thawed on ice and 1 ⁇ l or 5 ⁇ l of the ligation mixture reaction was added. The cells was incubated for 40 min on ice and then heat shocked by incubation at 42 °C for exactly 2 min.
  • Miniprep plasmid DNA was prepared from 5 ml cultures using a Biorobot 8000 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l of sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices). Aliquots of miniprep plasmid DNA (100-200 ng) were digested with BamHl for 2 h at 37 0 C and analysed by electrophoresis on 0.8% agarose gels.
  • Plasmids with inserts of the appropriate size were selected for DNA sequence analysis. Inserts were sequenced in both directions by mixing 200-500 ng plasmid DNA with either the T7 or T3 sequencing primers (see Table 3), and processed using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.
  • PCR products corresponding to INSP210 sequence were identified in several of the clones analysed but all appeared to contain a deletion of the sequence encoding exons 2 and 3.
  • the sequence of the cloned cDNA fragment is shown in Figure 6.
  • the plasmid containing the sequence verified full length INSP210SV2 PCR product is INSP210_pBSK.
  • Example 4 Construction of mammalian cell expression vectors for INSP210SV1 and INSP210SV2 Plasmids pCR4-TOPO-INSP210SVl ⁇ S117 and INSP210sv2_pBSK were used as PCR templates to generate pEAK12d and pDEST12.2 expression clones containing the ORF of INSP210SV1 and INSP210SV2 respectively with a 3' sequence encoding a C-terminal 6HIS tag, using the GatewayTM cloning methodology (Invitrogen).
  • Plasmid ⁇ CR4-TOPO-INSP21 OSVl-S 117 and Plasmid INSP210sv2jpBSK were used as PCR templates to generate the full - length ORF containing a C-terminal 6HIS tag and a stop codon.
  • the first stage of this Gateway cloning process involved a two step PCR reaction which generates the full - length ORF of INSP210SV1 or INSP210SV2 flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in-frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA).
  • 6HIS in-frame 6 histidine
  • the first PCR reaction PCRl (in a final volume of 50 ⁇ l) contains respectively: 1 ⁇ l (25 ng) of plasmid (pCR4-TOPO ⁇ INSP210SV1-S117 or INSP210sv2_pBSK), 4.0 ⁇ l dNTPs (10 niM), 5 ⁇ l of 1OX Pfx polymerase buffer, 1.5 ⁇ l MgSO4 (50 mM), 1.0 ⁇ l each of gene specific primer (to give a final concentration of 100 pico-moles) (INSP210 attB FP and INSP210 attB RP), and 0.5 ⁇ l Platinum Pfx DNA polymerase (Invitrogen).
  • the PCR reaction was performed using an initial denaturing step of 95 °C for 2 min, followed by 30 cycles of 94 °C for 30 s; 60 °C for 30 s and 68 °C for 1 min; and a final extension cycle of 68 °C for 5 minutes and a holding cycle of 4 °C.
  • the amplification products were directly purified using the Perfectprep Gel cleanup kit (Eppendorf) and recovered in 50 ⁇ l sterile water according to the manufacturer's instructions. A 2 ⁇ l aliquot was visualized on 0.8 % agarose gel in 1 X TAE buffer in order to verify that the products were of the expected molecular weight (486 bp for INSP210SV1 and 294 bp for INSP210SV2)
  • the second PCR reaction (in a final volume of 50 ⁇ l) contained 1 ⁇ l of diluted purified PCRl product (to a final concentration of 10 ng), 4.0 ⁇ l dNTPs (10 mM), 5 ⁇ l of 1OX Pfx polymerase buffer, 1.5 ⁇ l MgSO 4 (50 mM), 1.0 ⁇ l of each Gateway conversion primer (to give a final concentration of 100 picomoles) (ATTB PCR FP [unique] and ATTB PCR RP [unique]) and 0.5 ⁇ l of Platinum Pfx DNA polymerase.
  • the conditions for the 2nd PCR reaction were: 95 °C for 2 min, followed by 30 cycles of 94 °C for 30 s; 60 °C for 30 s and 68 0 C for 1 min; and a final extension cycle of 68 °C for 5 minutes and a holding cycle of 4 °C.
  • the PCR gel purified using the Perfectprep Gel cleanup kit (Eppendorf) and recovered in 50 ⁇ l sterile water according to the manufacturer's instructions.
  • the second stage of the Gateway cloning process involved sub cloning of the Gateway modified PCR products into the Gateway entry vector pDONR221 (Invitrogen) as follows: 5 ⁇ l of gel extracted products from each PCR2 were incubated with 1.5 ⁇ l pDONR221 vector (0.1 ⁇ g/ ⁇ l), 2 ⁇ l BP buffer and 1.5 ⁇ l of BP clonase enzyme mix (Invitrogen) in a final volume of 10 ⁇ l at RT for Ih. The reaction was stopped by addition of 1 ⁇ l proteinase K (2 ⁇ g/ ⁇ l) and incubated at 37 °C for a further 10 min.
  • pDONR221 Invitrogen
  • DH5 ⁇ strain (Invitrogen) was used to transform DH5 ⁇ strain (Invitrogen) as follows: a 50 ⁇ l aliquot of DH5 ⁇ cells was thawed on ice and 2 ⁇ l of reaction mixture added. The mixture was incubated for 30 min on ice and then heat shocked by incubation at 42 °C for exactly 45 s. Samples were returned to ice and 250 ⁇ l of warm SOC media (room temperature) was added. Samples were incubated with shaking (250 rpm) for 1 h at 37 °C. The transformation mixture was then plated on L-broth (LB) plates containing kanamycin (50 ⁇ g/ml) and incubated overnight at 37 0 C.
  • LB L-broth
  • the PCR mixture (in a final volume of 25 ⁇ l) contained 10 ⁇ l of the centrifuged cell lysate, 2.0 ⁇ l dNTPs (10 mM), 2.5 ⁇ l of Taq polymerase buffer, 0.5 ⁇ l of screening primers (to give a final concentration of 100 picomoles) (21M13 FP and ATTBl PCR RP [unique]) and 0.5 ⁇ l of Taq DNA polymerase.
  • the conditions for the screening PCR reaction were: 95 °C for 2 min, followed by 30 cycles of 94 °C for 30 s; 60 0 C for 30 s and 72 0 C for 1 min; and a final extension cycle of 72 0 C for 5 minutes and a holding cycle of 4 °C.
  • the PCR products were loaded onto a 0.8 % agarose gel to verify the fragment size.
  • Plasmid DNA 150-200 ng was subjected to DNA sequencing with 21M13 and M13Rev primers using the CEQ Dye Terminator Cycle sequencing Quick Start Kit (Beckman Coulter P/N 608120) according to the manufacturer's instructions. The primer sequences are shown in Table 3. Sequencing reactions were analyzed on CEQ 2000 XL DNA analysis system (Beckman Coulter P/N 608450).
  • Plasmid pDONR221_INSP210SVl - 6HIS contained the INSP210SV1-6HIS sequence and pDONR221_INSP210SV2 - 6HIS contained the INSP210SV2-6HIS.
  • 4.3 Sub cloning of Gateway compatible INSP210SV ORF into expression vectors pEAK12d and pDEST12.2
  • Plasmid DNA (2 ⁇ l or approx. 150 ng) of pDONR221_INSP210SVl-6HIS or pDONR221_INSP210SV2-6HIS were then used in a recombination reaction containing 1.5 ⁇ l of either pEAK12d vector or pDEST12.2 vector (0.1 ⁇ g / ⁇ l), 2 ⁇ l LR buffer and 1.5 ⁇ l of LR clonase (Invitrogen) in a final volume of 10 ⁇ l.
  • the reaction was stopped by addition of 1 ⁇ l proteinase K (2 ⁇ g/ ⁇ l) and incubated at 37 0 C for a further 10 min.
  • An aliquot of this reaction (2 ⁇ l) was used to transform DH5 ⁇ strain (Invitrogen) as follows: a 50 ⁇ l aliquot of DH5 ⁇ cells was thawed on ice and 2 ⁇ l of reaction mixture added. The mixture was incubated for 30 min on ice and then heat shocked by incubation at 42 0 C for exactly 45 s. Samples were returned to ice and 250 ⁇ l of warm SOC media (room temperature) was added. Samples were incubated with shaking (250 rpm) for 1 h at 37 °C.
  • the transformation mixture was then plated on L-broth (LB) plates containing Ampicillin (100 ⁇ g/ml) and incubated overnight at 37 0 C.
  • Five transformants from each LR reaction were picked and patched on LB agar plates containing Ampicillin (100 ⁇ g/ml) and incubated overnight at 37 0 C.
  • a scoop of the grown culture from the patched plate was resuspended in 50 ⁇ l of water and boiled for 5 minutes to lyse the cells. The cell lysate was centrifuged to remove the cell debris and the supernatant obtained was used as a template for colony PCR screening.
  • the PCR mixture (in a final volume of 25 ⁇ l) contained 10 ⁇ l of the centrifuged cell lysate, 2.0 ⁇ l dNTPs (10 mM), 2.5 ⁇ l of Taq polymerase buffer, 0.5 ⁇ l of screening primers (to give a final concentration of 100 picomoles and 0.5 ⁇ l of Taq DNA polymerase.
  • pEAK12d clones were screened using the primers pEAK12 FP and pEAK12 RP and pDEST12.2 clones were screened using the primers 21M13FP and M13Rev RP.
  • the conditions for the screening PCR reaction were: 95 °C for 2 min, followed by 30 cycles of 94 °C for 30 s; 6O 0 C for 30 s and 72 0 C for 1 min; and a final extension cycle of 72 0 C for 5 minutes and a holding cycle of 4 0 C.
  • the PCR products were loaded onto a 0.8 % agarose gel to verify the fragment size.
  • Plasmid DNA (150 - 200 ng) in the pEAK12d vector was subjected to DNA sequencing with the sequencing primers pEAK12 FP and pEAK12 RP as described above.
  • Plasmid DNA (150 - 200 ng) in the pDEST12.2 vector was subjected to DNA sequencing with the sequencing primers 21M13 FP and M13Rev RP as described above.
  • Maxi-prep DNA was prepared from a 500 ml culture of the sequence verified clones (pEAK12d_INSP210SVl-6HIS and pEAK12d_INSP210SV2-6HIS) using a Qiagen mega plasmid prep kit (cat no. 12183). Plasmid DNA was resuspended at a minimum concentration of 1 ⁇ g/ ⁇ l in sterile water (or 10 mM Tris-HCl pH 8.5) and stored at -20 0 C.
  • Endotoxin-free maxi-prep DNA was prepared from a 500 ml culture of the sequence verified clones (pDEST12.2_INSP210SVl-6HIS and pDEST12.2_INSP210SV2-6HIS) using the EndoFree Plasmid Mega kit (Qiagen cat no. 12381) according to the manufacturer's instructions. Purified plasmid DNA was resuspended in endotoxin free TE buffer at a final concentration of at least 3 ⁇ g/ ⁇ l and stored at -20 °C.
  • Example 5 Microarray studies
  • 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.”
  • Probe Synthesis Methodologies were carried out according to Agilent instructions. Essentially, 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).
  • INSP210 is formed from separate component exons. We intend to profile the chips using probe synthesized from 10 normal tissues, bone marrow, brain, lung, ovary, PBMCs, placenta, prostate, spleen and testis. Expression reports are obtainable on an exon by exon basis.
  • Averaging is performed for the data, using the One-step Tukey Bi-Weight Algorithm (Data Analysis and Regression: A Second Course in Statistics", Mosteller and Tukey, Addison- Wesley, 1977, pp. 203-209; see also Affymetrix MAS5.0 algorithm).
  • Tukey Bi-Weight Algorithm Data Analysis and Regression: A Second Course in Statistics", Mosteller and Tukey, Addison- Wesley, 1977, pp. 203-209; see also Affymetrix MAS5.0 algorithm.
  • the purpose of this is to define a robust estimate of the average value of a dataset. In this case our datasets will comprise multiple probe expression values for a single exon.
  • This custom array is useful for a number of reasons. First, it allows the existence and sequence of the transcript to be confirmed. Second, the tissue distribution of the INSP210 polypeptide sequence can be evaluated and thus the role of this polypeptide in disease can be clarified. The array can also be used as a diagnostic tool, to diagnose disease incidence in patients with disease conditions with which this polypeptide is correlated. The use of exon-specific probes allows any variance in expression of splice variants of this polypeptide sequence to be evaluated, in general, in specific tissues and in specific disease states.

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Abstract

L'invention concerne de nouvelles protéines, appelées INSP210 et INSP211, identifiées comme des protéines à domaine(s) NTR de type nétrine, ainsi que l'utilisation de ces protéines et séquences d'acides nucléiques des gènes codants dans le diagnostic, la prévention et le traitement de maladies.
PCT/GB2006/004376 2005-11-23 2006-11-23 Proteine a domaine(s) ntr de type netrine WO2007060431A1 (fr)

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GB0523827A GB0523827D0 (en) 2005-11-23 2005-11-23 Netrin-like (NTR) domain containing protein
GB0523827.4 2005-11-23

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078960A2 (fr) * 1999-06-23 2000-12-28 Corixa Corporation Compositions et methodes destinees au traitement et au diagnostic du cancer du sein
CN1352017A (zh) * 2000-11-06 2002-06-05 上海博德基因开发有限公司 一种新的多肽——α－1抑制子III－10.67和编码这种多肽的多核苷酸

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078960A2 (fr) * 1999-06-23 2000-12-28 Corixa Corporation Compositions et methodes destinees au traitement et au diagnostic du cancer du sein
CN1352017A (zh) * 2000-11-06 2002-06-05 上海博德基因开发有限公司 一种新的多肽——α－1抑制子III－10.67和编码这种多肽的多核苷酸

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE EBI EMBL; 4 April 2003 (2003-04-04), BODE GENE DEV CO LTD SHANGHAI: "Alpha-1 inhibitor III-10.67 amino acid sequence", XP002423166, Database accession no. ABP60093 *

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