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WO2004029295A1 - Methode de diagnostic du cancer - Google Patents

Methode de diagnostic du cancer Download PDF

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Publication number
WO2004029295A1
WO2004029295A1 PCT/SG2003/000228 SG0300228W WO2004029295A1 WO 2004029295 A1 WO2004029295 A1 WO 2004029295A1 SG 0300228 W SG0300228 W SG 0300228W WO 2004029295 A1 WO2004029295 A1 WO 2004029295A1
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Prior art keywords
sprouty2
sequence
expression
cell
nucleic acid
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Graeme Roy Guy
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • This invention relates to the field of medicine.
  • it relates to treatment and diagnosis of diseases, in particular breast cancer, as well as compositions for such use.
  • breast carcinoma is the second leading cause of cancer-related death in women (18).
  • Breast cancer tops the cancer list for women in Singapore, with 700-800 new cases being diagnosed each year (19).
  • 180,000 women are diagnosed annually with new cases of breast cancer (18).
  • better diagnosis and routine screening around a quarter of the cases will die from their disease.
  • the BRCA1 and BRCA2 genes involved in hereditary breast cancers do not appear to play a role in sporadic cases, which represent by far the majority of the cases.
  • Amplification or over-expression of oncogenes [c-myc, erbB2, cyclin Dl and epidermal growth factor receptor (EGF-R)] (20, 21) and loss of tumour suppressor genes [p53, PTEN, PTCH (patched), MKK4] (22-25) occur in only a fraction of the cases.
  • GEF-R epidermal growth factor receptor
  • p53, PTEN, PTCH (patched), MKK4] (22-25) occur in only a fraction of the cases.
  • Recently a gene encoding a novel cytokine, HIN-1 was identified via SAGE (Serial Analysis of Gene Expression) as a candidate breast tumour suppressor gene that is not expressed and is hypermethylated in the majority of breast cancers (26).
  • HIN-1 is inactivated in pre- invasive tumours, such as DCIS and LCIS, and its methylation is high (over 70%) in early- stage tumours, which makes it a very good marker for early detection of breast cancer.
  • pre- invasive tumours such as DCIS and LCIS
  • methylation is high (over 70%) in early- stage tumours, which makes it a very good marker for early detection of breast cancer.
  • Several other genes have been demonstrated to be hypermethylated in breast carcinomas, including; pi 6, E-cadherin, BRCA1, oestrogen receptor, GSTP1 (glutathione S-transferase PI), MDGI (mammary-derived growth factor inhibitor), HoxA5 and 14-3-3- ⁇ (27-34) .
  • relatively low numbers of tumours were ranked positive for hypermethylation of these genes with the exception ofl4-3-3- ⁇ that has abnormal methylation levels in around 50% of invasive carcinomas.
  • the silencing of HIN-1 is however not specific to breast tumour
  • markers for breast cancer detection and in particular, those which are tissue specific for breast tissue.
  • a method of diagnosis of a cancer in an individual comprising detecting modulation of expression of a Sprouty2 sequence in the individual, or any part of the individual.
  • the cancer comprises breast cancer.
  • the method comprises detecting down-regulation of Sprouty2 expression in a breast cell or tissue of or from the individual.
  • a Sprouty2 nucleic acid is detected by means of a probe comprising at least a portion of a nucleic acid having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.
  • the method comprises detecting a Sprouty2 polypeptide, preferably by means of an antibody to Sprouty2, in a sample comprising a breast cell or tissue from the individual.
  • the expression of Sprouty2 in the sample is compared to the expression of Sprouty2 in a control breast cell known to be non-cancerous.
  • a down-regulation of Sprouty2 expression in the sample compared to the control breast cell is diagnostic of breast cancer, or susceptibility to breast cancer.
  • a method for identifying a pre-cancerous breast cell comprising detecting a reduced level of a Sprouty2 polypeptide and/or a Sprouty2 nucleic acid in the cell, or an extract thereof.
  • a specific binding agent for Sprouty2 for use in a method of diagnosis of a cancer, preferably breast cancer.
  • the specific binding agent may comprise: (a) a nucleic acid probe comprising a Sprouty2 nucleic acid, or a fragment thereof capable of hybridisation to a Sprouty2 sequence; (b) a nucleic acid probe having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof; (c) a primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably having a sequence selected from the sequences set out in Tables 1 A or IB; (d) a pair of primers comprising a forward primer selected from sequences depicted in Table 1A together with a reverse primer selected from sequences depicted in Table IB; and (e) an anti-Sprouty2 antibody.
  • a method of treatment or prophylaxis of a cancer in an individual comprising modulating the amount of a Sprouty2 polypeptide or nucleic acid in a cell of an individual.
  • the cancer comprises a breast cancer.
  • the amount of a Sprouty2 polypeptide or nucleic acid is increased in a breast cell, preferably a diseased breast cell, of the individual.
  • the amount of Sprouty2 polypeptide or nucleic acid is increased specifically in a breast cell of the individual.
  • the amount of a Sprouty2 polypeptide or nucleic acid is not substantially increased in any other cell or tissue type.
  • the amount of Sprouty2 polypeptide or nucleic acid is preferably not substantially increased in any other normal cell or tissue, preferably of the same individual.
  • a method of manipulating a cell comprising the steps of: (a) detecting a reduced level of a Sprouty2 polypeptide or nucleic acid in a cell, or an extract thereof; (b) increasing the level of a Sprouty2 polypeptide or nucleic acid in the cell.
  • the cell is derived from or present in an individual at risk of developing breast cancer.
  • the expression or activity of an endogenous Sprouty2 sequence is up-regulated.
  • a control sequence preferably a promoter and/or an enhancer sequence of Sprouty2 is replaced with an endogenous control sequence.
  • the expression of a Sprouty2 sequence is up-regulated in a breast cell of the individual, but not substantially in any other cell or tissue type.
  • an expression construct capable of delivering breast cell specific expression of Sprouty2 is introduced into a cell of the individual.
  • the expression construct comprises a nucleic acid according to the sixth aspect of the invention.
  • the present invention in a sixth aspect, provides a nucleic acid construct comprising a Sprouty2 gene or a coding portion thereof, together with one or more control elements selected from the group consisting of: (a) a tumour specific promoter selected from the group consisting of: vascular endothelial growth factor (VEGF) promoter, vascular endothelial growth factor receptor- 1 (NEGFR-1) promoter, VEGFR-2 promoter, c-erbBl promoter, L-plastin promoter, Bcl-2 promoter and MUCl promoter; (b) a breast tissue specific promoter selected from the group consisting of: human ⁇ -lactalbumin (ALA) promoter, ovine ⁇ -lactoglobulin (BLG) promoter and a long terminal repeat (LTR) of a mouse mammary tumour virus (MMTV); (c) an inducible promoter selected from the group consisting of: a stress gene promoter, a heat shock protein (HSP) promoter and a multidrug
  • a host cell comprising a nucleic acid construct according to the sixth aspect of the invention.
  • transgenic non-human animal comprising a transgene which does not express, or expresses a reduced level, of Sprouty2.
  • transgenic non-human animal according the ninth aspect of the invention use of a transgenic non-human animal according the ninth aspect of the invention as a model for breast cancer.
  • we provide a method of identifying a molecule capable of binding to a Sprouty2 polypeptide comprising contacting a Sprouty2 polypeptide with a candidate molecule and determining whether the candidate molecule binds to the Sprouty2 polypeptide.
  • a method of identifying a modulator of Sprouty2 expression comprising contacting a cell with a candidate molecule, and detecting elevated expression of Sprouty2 in or of the cell.
  • a method of identifying a drug comprising exposing a transgenic animal according to the ninth aspect of the invention to a candidate molecule, and monitoring the development of a breast cancer in the transgenic animal.
  • the methods preferably further comprise isolating and/or synthesising the molecule.
  • a fourteenth aspect of the present invention a molecule identified, isolated or synthesised by a method according to the eleventh, twelfth or thirteenth aspect of the invention.
  • a sixteenth aspect of the present invention we provide a molecule according to the fourteenth aspect of the invention for use in a method of treatment of a breast cancer.
  • nucleic acid having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof capable of specifically hybridising to a Sprouty2 sequence.
  • a primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably having a sequence selected from the sequences set out in Tables 1A or IB.
  • the Sprouty2 comprises human Sprouty2.
  • this comprises a human Sprouty2 nucleic acid having a GenBank accession number NM_005842 or AF039843, or a human S ⁇ routy2 polypeptide having a GenBank accession number 043597.
  • Figure l In order to verify that the [ ⁇ - 32 P] dCTP-labeled, N-terminal hS ⁇ ry2 cDNA probe is specific for the hSpry2 isoform, a dot blot consisting of full length cDNA of hSpryl, hSpry2, hSpry3 and hSpry4 is prepared. The blots are probed with each radiolabelled N-terminal hSpry2 cDNA. Rows: Full length cDNA. Columns: 32 P labelled N-terminal hSpry cDNA probe. Row 1: hSpryl, Row 2: hSpry2, Row 3: hSpry3, Row 4: hSpry4. Column 1: hSpryl, Column 2: hSpry2, Column 3: hSpry3, Column 4: hSpry4.
  • FIG. 2A cDNAs are immobilized on the commercial Cancer Profiling array as supplied by Clontech.
  • Figure 2B An autoradiograph showing binding of the specific, radiolabelled hS ⁇ ry2 probe to the Cancer Profiling Array.
  • the X-ray film is exposed to the blot for 10 hours.
  • FIG. 2C-2E A scatter-plot diagram of the relationship between the expression of hS ⁇ ry2 in normal compared with tumour tissue. Each spot is measured on a densitometer, background levels are subtracted and the ratio of change (density of normal spot/density of tumour spot expressed in arbitrary units) is plotted on a scatter diagram. The midpoint line is indicative of no change (ie 1:1 ratio). The dotted lines either side of the midpoint line represents an estimate of experimental error and/or insignificant change based on an analysis of micro-array data. Spots under and over the line represent decreased and increased expression of hSpry2, respectively, in tumour tissues compared with normal tissue from the same patient. The ratio of metastasic tumours are indicated in pink diamonds.
  • X-axis normal tissue (arbitrary units);
  • Y-axis tumour tissue (arbitrary units).
  • Figure 2D A scatter diagram comparing the expression ratios (normal/tumour) of hSpry2 in breast versus colon tumour tissues.
  • X-axis normal tissue (arbitrary units); Y-axis: tumour tissue (arbitrary units).
  • Figure 2E A scatter diagram comparing the expression ratios (normal/tumour) of hS ⁇ ry2 in breast versus uterine tumour tissues.
  • X-axis normal tissue (arbitrary units);
  • Y-axis tumour tissue (arbitrary units).
  • Figure 2F A re-probed Cancer Profiling Array blot to demonstrate equality of cDNA loading.
  • the blot shown in Figure 2B is stripped and incubated with a [ - 3 P] dCTP -labeled ubiquitin probe to reveal equality of loading of the individual cDNAs.
  • SEQ D3 NO: 1 shows the nucleic acid sequence of a Homo sapiens Sprouty2 fragment, comprising N-terminal sequences.
  • SEQ H) NO: 2 shows the amino acid sequence of a Homo sapiens Sprouty2 fragment, comprising N-terminal sequences.
  • SEQ ID NO: 3 shows the nucleic acid sequence of full length Homo sapiens Sprouty2.
  • SEQ ID NO: 4 shows the amino acid sequence of full length Homo sapiens Sprouty2.
  • Sprouty and “Spry” should be taken to refer to any Sprouty sequence, including a Sprouty protein or a Sprouty nucleic acid, including any member of the Sprouty family such as Sprouty 1, Sprouty2, Sprouty3 and Sprouty4 and any fragment, variant homologue, derivative, variant thereof.
  • Sprouty2 and “Spry2” should preferably be taken to refer to any Sprouty2 sequence such as a Sprouty2 polypeptide, Sprouty2 nucleic acid, fragment, derivative, homologue or variant, "human Sprouty2” and “hSpry2” should be taken to be synonymous with each other, and to refer to any human Sprouty2 sequence.
  • Sprouty2 The properties and activities of Sprouty2 are described in, for example, in refs 1 to 12.
  • the term "Sprouty2 polypeptide” is intended to refer to any one or more of Sprouty 2 (Homo sapiens) having GenBank accession number AAC04258 ; Sprouty-2 (Mus musculus) having GenBank accession number AAD34167 ; Sprouty 2 (Gallus gallus) having GenBank accession number AAD56005 ; Sprouty 2 (Mus musculus) having GenBank accession number AAD56006 ; Sprouty homologue 2 (Homo sapiens) having GenBank accession number AAHl 5745 ; Sprouty homologue 2 (Mus musculus) having GenBank accession number NP_036027 ; Sprouty homologue 2 (Spry- 2) from Gallus gallus having GenBank accession number Q9PTL2 ; Sprouty homologue 2 (Spry-2) from Mus musculus having GenBank accession number Q9QXV8 ; Sprouty 2 (Xenopus
  • human Sprouty2 “hSprouty2” and “hSpry2” should be taken to be synonymous, and in the context of polypeptides, to refer to any human Sprouty2 sequence, including but not limited to: Sprouty 2 (Homo sapiens) having GenBank accession number AAC04258; Sprouty homologue 2 (Homo sapiens) having GenBank accession number AAHl 5745; Sprouty homologue 2 (Homo sapiens) having GenBank accession number NP_005833; and Sprouty homologue 2 (Spry-2) from Homo sapiens having GenBank accession number 043597.
  • a "Sprouty2 polypeptide” comprises or consists of a human Sprouty2 polypeptide, preferably, the sequence having accession number 043597 and labelled “Sprouty homolog 2 (Spry-2)".
  • human S ⁇ routy2 comprises or preferably has the sequence shown in SEQ ID NO: 4.
  • polypeptide refers to 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.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
  • Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-inking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-inks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • polypeptide includes the various synthetic peptide variations known in the art, such as a retroinverso D peptides.
  • the peptide may be an antigenic determinant and/or a T-cell epitope.
  • the peptide may be immunogenic in vivo.
  • the peptide is capable of inducing neutralising antibodies in vivo.
  • the resultant amino acid sequence has one or more activities, preferably, biological activities in common with a Sprouty2 polypeptide, preferably a human Sprouty2 polypeptide.
  • a Sprouty2 homologue may have a lowered expression level in breast cancer cells compared to normal breast cells.
  • the term "homologue” covers identity with respect to structure and/or function providing the resultant amino acid sequence has Sprouty2 activity.
  • sequence identity i.e. similarity
  • sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
  • such activities may include any one or more of the following: ability to inhibit one or more events downstream of receptor tyrosine kinases; ability to prevent activation of Ras/Raf; inhibition of the Ras/Raf/MAPK pathway; ability to downregulate ERK phosphorylation when the Ras/MAPK pathway is activated.
  • Sprouty2 variants, homologues, derivatives and fragments are also of use in the methods and compositions described here.
  • variants include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to a sequence.
  • references to “Sprouty2” includes references to such variants, homologues, derivatives and fragments of Sprouty2.
  • a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • an “insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring substance.
  • substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
  • Sprouty2 polypeptides as described here may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent amino acid sequence. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • Sprouty2 polypeptides may further comprise heterologous amino acid sequences, typically at the N-terminus or C-terminus, preferably the N-terminus.
  • Heterologous sequences may include sequences that affect intra or extracellular protein targeting (such as leader sequences).
  • Heterologous sequences may also include sequences that increase the immunogenicity of the Sprouty2 polypeptide and/or which facilitate identification, extraction and/or purification of the polypeptides.
  • Another heterologous sequence that is particularly preferred is a polyamino acid sequence such as polyhistidine which is preferably N-terminal.
  • a polyhistidine sequence of at least 10 amino acids, preferably at least 17 amino acids but fewer than 50 amino acids is especially preferred.
  • the Sprouty2 polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • Sprouty2 polypeptides as described here are advantageously made by recombinant means, using known techniques. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Such polypeptides may also be produced as fusion proteins, for example to aid in extraction and purification.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ - galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site.
  • the fusion protein will not hinder the function of the protein of interest sequence.
  • the Sprouty2 polypeptides may be in a substantially isolated form. This term is intended to refer to alteration by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide, nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide, nucleic acid or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
  • Sprouty2 protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
  • a Sprouty2 polypeptide may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, for example, 95%, 98% or 99% of the protein in the preparation is a Sprouty2 polypeptide.
  • the Sprouty2 polypeptides may therefore comprise a sequence which corresponds to at least part of a homologous region.
  • a homologous region shows a high degree of homology between at least two species.
  • the homologous region may show at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identity at the amino acid level using the tests described above.
  • Peptides which comprise a sequence which corresponds to a homologous region may be used in therapeutic strategies as explained in further detail below.
  • the Sprouty2 peptide may comprise a sequence which corresponds to at least part of a heterologous region.
  • a heterologous region shows a low degree of homology between at least two species.
  • the N-terminus of Sprouty is known to be heterologous between Sproutyl, S ⁇ routy2, Sprouty3 and Sprouty4.
  • the Sprouty2 polypeptides disclosed for use include homologous sequences obtained from any source, for example related viral bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
  • polypeptides also include those encoding homologues of Sprouty2 from other species including ammals such as mammals (e.g. mice, rats or rabbits), especially primates, more especially humans. More specifically, homologues include human homologues.
  • a homologous sequence is taken to include an amino acid sequence which is at least 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level, preferably over at least 50 or 100, preferably 200, 300, 400 or 500 amino acids with the sequence of a relevant Sprouty2 sequence.
  • homology should typically be considered with respect to those regions of the sequence known to be essential for protein function rather than non- essential neighbouring sequences. This is especially important when considering homologous sequences from distantly related organisms.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present document it is preferred to express homology in terms of sequence identity.
  • homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate % identity between two or more sequences.
  • % identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid- Chapter 18), FASTA (Altschul et al, 1990, J. Mol.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • variant or derivative in relation to amino acid sequences includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence retains substantially the same activity as the unmodified sequence, preferably having at least the same activity as the Sprouty2 polypeptides (e.g., asequence having accession number 043597 and labelled “Sprouty homolog 2 (Spry-2)".).
  • Polypeptides having the Sprouty2 amino acid sequence disclosed here, or fragments or homologues thereof may be modified for use in the methods and compositions described here. Typically, modifications are made that maintain the biological activity of the sequence.
  • Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the biological activity of the unmodified sequence. Alternatively, modifications may be made to deliberately inactivate one or more functional domains of the polypeptides described here. Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.
  • Polypeptides for use in the methods and compositions described here also include fragments of the full length sequence of any of the Sprouty2 polypeptides identified above. Preferably fragments comprise at least one epitope. Methods of identifying epitopes are well known in the art. Fragments will typically comprise at least 6 amino acids, more preferably at least 10, 20, 30, 50 or 100 amino acids.
  • fragments comprising, preferably consisting of, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 ; 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,
  • peptides comprising a portion of a Sprouty2 polypeptide as described here.
  • fragments of Sprouty2 and its homologues, variants or derivatives are included.
  • the peptides may be between 2 and 200 amino acids, preferably between 4 and 40 amino acids in length.
  • the peptide may be derived from a Sprouty2 polypeptide as disclosed here, for example by digestion with a suitable enzyme, such as trypsin.
  • the peptide, fragment, etc may be made by recombinant means, or synthesised synthetically.
  • a preferred fragment of Sprouty2 comprises a human Sprouty2 fragment, for example, a fragment of a sequence having accession number 043597.
  • a preferred fragment of Sprouty2 may preferably comprise an N-terminal sequence of human Sprouty2, such as a sequence shown in SEQ ID NO: 2.
  • Such N-terminal fragments of Sprouty2 are particularly useful as the sequence similarity at the N-terminus between Sprouty 1, S ⁇ routy2, Sprouty3 and Sprouty4 is low. Accordingly, fragments comprising N- terminal sequences may be used to generate probes to preferentially detect Sprouty2 expression, for example, through antibodies generated against such fragments. These antibodies would be expected to bind specifically to Sprouty2, and are useful in the methods of diagnosis disclosed here.
  • Particularly preferred fragments include a S ⁇ routy2 fragment sequence depicted as
  • SEQ ID NO: 2 SEQ ID NO: 2. It will be appreciated that a number of residues C-terminal side of the SEQ ID NO: 2 sequence may be included without influencing the specificity.
  • Sprouty2 and its fragments, homologues, variants and derivatives may be made by recombinant means. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis.
  • the proteins may also be produced as fusion proteins, for example to aid in extraction and purification.
  • fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the function of the protein of interest sequence.
  • Proteins may also be obtained by purification of cell extracts from animal cells.
  • the Sprouty2 polypeptides, variants, homologues, fragments and derivatives disclosed here may be in a substantially isolated form. It will be understood that such polypeptides may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
  • a Sprouty2 variant, homologue, fragment or derivative may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein.
  • the Sprouty2 polypeptides, variants, homologues, fragments and derivatives disclosed here may be labelled with a revealing label.
  • the revealing label may be any suitable label which allows the polypeptide , etc to be detected. Suitable labels include radioisotopes, e.g. I, enzymes, antibodies, polynucleotides and linkers such as biotin. Labelled polypeptides may be used in diagnostic procedures such as immunoassays to determine the amount of a polypeptide in a sample. Polypeptides or labelled polypeptides may also be used in serological or cell-mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols.
  • a Sprouty2 polypeptides, variants, homologues, fragments and derivatives disclosed here, optionally labelled, may also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick.
  • Such labelled and/or immobilised polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
  • Such polypeptides and kits may be used in methods of detection of antibodies to the polypeptides or their allelic or species variants by immunoassay.
  • Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed.
  • the Sprouty2 polypeptides, variants, homologues, fragments and derivatives disclosed here may be used in in vitro or in vivo cell culture systems to study the role of their corresponding genes and homologues thereof in cell function, including their function in disease.
  • truncated or modified polypeptides may be introduced into a cell to disrupt the normal functions which occur in the cell.
  • the polypeptides may be introduced into the cell by in situ expression of the polypeptide from a recombinant expression vector (see below).
  • the expression vector optionally carries an inducible promoter to control the expression of the polypeptide.
  • host cells such as insect cells or mammalian cells
  • post-translational modifications e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
  • Such cell culture systems in which the Sprouty2 polypeptides, variants, homologues, fragments and derivatives disclosed here are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides in the cell.
  • the methods and compositions described here may employ, as a means for detecting expression levels of Sprouty2, Sprouty2 polynucleotides, Sprouty2 nucleotides and Sprouty2 nucleic acids, as well as variants, homologues, derivatives and fragments of any of these.
  • Sprouty2 fragments useful for the methods of diagnosis described here.
  • the Sprouty2 nucleic acids may also be used for the methods of treatment or prophylaxis described.
  • Sprouty2 polynucleotide may be used interchangeably, and should be understood to specifically include both cDNA and genomic S ⁇ routy2 sequences. These terms are also intended to include a nucleic acid sequence capable of encoding a Sprouty2 polypeptide and/or a fragment, derivative, homologue or variant of this. Where reference is made to a Sprouty2 nucleic acid, this should be taken as a reference to any member of the Sprouty2 family of nucleic acids.
  • Sprouty2 nucleic acids selected from the group consisting of: fk66hl l.xl Zebrafish Research Genetics C32 Gn Danio rerio cDNA 3' similar to TR:O43597 043597 SPROUTY 2 mRNA sequence having accession number BE016191; fk93el l.xl Zebrafish Research Genetics C32 fin Danio rerio cDNA 3' similar to TR:O43597 043597 SPROUTY 2 mRNA sequence; Gallus gallus sprouty 2 (Spry2) mRNA, complete eds having accession numberAF 176904 ; Homo sapiens chromosome 13 working draft sequence segment having accession number NT_024524; Homo sapiens Sprouty 2 (SPRY2) mRNA, complete eds having accession number AF039843; Homo sapiens sprouty homolog 2 (Drosophila) (SPRY2), mRNA having accession number mRNA having
  • a Sprouty2 nucleic acid comprises a human Sprouty2 (hSpry2) sequence selected from the group consisting of: Homo sapiens chromosome 13 working draft sequence segment having accession number NT_024524; Homo sapiens Sprouty 2 (SPRY2) mRNA, complete eds having accession number AF039843; Homo sapiens sprouty homolog 2 (Drosophila) (SPRY2), mRNA having accession number NM__005842; Homo sapiens, sprouty (Drosophila) homolog 2, clone IMAGE:3533337, mRNA having accession number BC004205; and Homo sapiens, sprouty (Drosophila) homolog 2, clone MGC:23039 IMAGE:4863701, mRNA, complete eds having accession number BCO 15745.
  • hSpry2 human Sprouty2 sequence selected from the group consisting of: Homo sapiens chromosome
  • a Sprouty2 nucleic acid comprises or consists of a human Sprouty2 nucleic acid, preferably, the sequence having accession number NM )05842 and denoted “Homo sapiens sprouty homolog 2 (Drosophila) (SPRY2), mRNA” or a sequence having accession number AF039843 and denoted "Homo sapiens Sprouty 2 (SPRY2) mRNA, complete eds”.
  • a Sprouty2 nucleic acid comprises or has a sequence shown in SEQ ID NO: 3.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • nucleotide sequence refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
  • the term nucleotide sequence may be prepared by use of recombinant DNA techniques (for example, recombinant DNA).
  • nucleotide sequence means DNA.
  • Other Nucleic Acids are examples of nucleic Acids
  • nucleic acids which are fragments, homologues, variants or derivatives of Sprouty2 nucleic acids.
  • variant variant
  • homologue derivative
  • fragment fragment
  • references to “S ⁇ routy2” and “Sprouty2” include references to such variants, homologues, derivatives and fragments of Sprouty2.
  • the resultant nucleotide sequence encodes a polypeptide having any one or more Sprouty2 activity.
  • the term "homologue” is intended to cover identity with respect to structure and/or function such that the resultant nucleotide sequence encodes a polypeptide which has Sprouty2 activity.
  • a homologue etc of Sprouty2 may have a reduced expression level in breast cancer cells compared to normal breast cells.
  • sequence identity i.e. similarity
  • sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity.
  • sequence identity there is at least 95%, more preferably at least 98%, sequence identity to a relevant sequence (e.g., a human Sprouty2 sequence having GenBank accession number NM_005842 or a sequence having GenBank accession number AF039843). These terms also encompass allelic variations of the sequences.
  • a relevant sequence e.g., a human Sprouty2 sequence having GenBank accession number NM_005842 or a sequence having GenBank accession number AF039843.
  • Sprouty2 nucleic acid variants, fragments, derivatives and homologues may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of this document, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • both strands of the duplex are encompassed by the methods and compositions described here.
  • the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included.
  • variants in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • variant, homologues or derivatives code for a polypeptide having biological activity.
  • fragments, homologues, variants and derivatives of Sprouty2 comprise modulated activity, as set out above.
  • a "homologue” has preferably at least 5% identity, at least 10% identity, at least 15% identity, at least 20% identity, at least 25% identity, at least 30% identity, at least 35% identity, at least 40% identity, at least 45% identity, at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70%> identity, at least 75%> identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to the relevant sequence (e.g., a human Sprouty2 sequence having GenBank accession number NM_005842 or a sequence having GenBank accession number AF039843).
  • relevant sequence e.g., a human Sprouty2 sequence having GenBank accession number NM_005842 or a sequence having GenBank accession number AF039843.
  • nucleotide identity comparisons may be conducted as described above.
  • a preferred sequence comparison program is the GCG Wisconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch.
  • the default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.
  • nucleotide sequences that are capable of hybridising selectively to any of the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction technologies.
  • Polynucleotides capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement may be at least 40% homologous, at least 45% homologous, at least 50% homologous, at least 55% homologous, at least 60% homologous, at least 65%> homologous, at least 70% homologous, at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, or at least 95% homologous to the corresponding nucleotide sequences presented herein (e.g., a human Sprouty2 sequence having GenBank accession number NM_005842 or a sequence having GenBank accession number AF039843).
  • such polynucleotides will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • the term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide is found to hybridize to the probe at a level significantly above background.
  • the background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening.
  • background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P or 33 P or with non-radioactive probes (e.g., fluorescent dyes, biotin or digoxigenin).
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
  • Polynucleotides which are not 100% identical to the prefe ⁇ ed sequences e.g., a human Sprouty2 sequence having GenBank accession number NM_005842 or a sequence having GenBank accession number AF039843
  • homologues, variants and derivatives of Sprouty2 can be obtained in a number of ways.
  • Other variants of the sequences may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • Sprouty2 homologues may be identified from other individuals, or other species.
  • Further recombinant Sprouty2 nucleic acids and polypeptides may be produced by identifying co ⁇ esponding positions in the homologues, and synthesising or producing the molecule as described elsewhere in this document.
  • homologues of S ⁇ routy2 particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to human Sprouty2.
  • Such homologues may be used to design non- human Sprouty2 nucleic acids, fragments, variants and homologues. Mutagenesis may be carried out by means known in the art to produce further variety.
  • Sequences of Sprouty2 homologues may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any of the Sprouty2 nucleic acids, fragments, variants and homologues, or other fragments of Sprouty2 under conditions of medium to high stringency.
  • variants and allelic variants of the polypeptide or nucleotide sequences disclosed here may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the Sprouty2 nucleic acids.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. It will be appreciated by the skilled person that overall nucleotide homology between sequences from distantly related organisms is likely to be very low and thus in these situations degenerate PCR may be the method of choice rather than screening libraries with labelled fragments the Sprouty2 sequences.
  • homologous sequences may be identified by searching nucleotide and/or protein databases using search algorithms such as the BLAST suite of programs.
  • polynucleotides may be obtained by site directed mutagenesis of characterised sequences, for example, Sprouty2 nucleic acids, or variants, homologues, derivatives or fragments thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • the polynucleotides described here may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • Such primers, probes and other fragments will be at least 8, 9, 10, or 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term "polynucleotides" as used herein.
  • Polynucleotides such as a DNA polynucleotides and probes may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Primers comprising fragments of Sprouty2 are particularly useful in the methods of detection of Sprouty2 expression, preferably down-regulation of Sprouty2 expression, for example, as associated with breast cancer.
  • Suitable primers for amplification of Sprouty2 may be generated from any suitable stretch of the 949 nucleotide bases of Sprouty2.
  • Particularly prefe ⁇ ed primers are those capable of amplifying a sequence of Sprouty2 which is specific, i.e., does not have significant homology to other Sprouty family members such as Sprouty 1, Sprouty3 and Sprouty4.
  • Such primers preferably are capable of binding to and amplifying an "N-terminal" region of Sprouty2.
  • a primer for amplification of Sprouty2 may comprise a sequence 5' ATGGAGGCCAGAGCTCAGAGT 3 ' or a sequence 5' CCTGTAGGCGTGCAGGCC 3'.
  • a primer pair comprising the two preceding sequences is provided.
  • Sprouty2 primers may be provided on their own, they are most usefully provided as primer pairs, comprising a forward primer and a reverse primer.
  • a Sprouty2 forward primer comprises a sequence selected from the group consisting of the residues of SEQ ID NO: 1 shown in Table 1 A below:
  • a Sprouty2 reverse primer comprises a sequence selected from the group consisting of the residues of SEQ ID NO: 1 shown in Table IB below:
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides), bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector
  • Polynucleotides or primers may carry a revealing label. Suitable labels include radioisotopes such as 32 P or 35 S, digoxigenin, fluorescent dyes, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers and may be detected using by techniques known per se. Polynucleotides or primers or fragments thereof labelled or unlabeled may be used by a person skilled in the art in nucleic acid-based tests for detecting or sequencing polynucleotides in the human or animal body.
  • Such tests for detecting generally comprise bringing a biological sample containing DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and detecting any duplex formed between the probe and nucleic acid in the sample.
  • detection may be achieved using techniques such as PCR or by immobilising the probe on a solid support, removing nucleic acid in the sample which is not hybridised to the probe, and then detecting nucleic acid which has hybridised to the probe.
  • the sample nucleic acid may be immobilised on a solid support, and the amount of probe bound to such a support can be detected. Suitable assay methods of this and other formats can be found in for example WO89/03891 and WO90/13667.
  • Tests for sequencing nucleotides involve bringing a biological sample containing target DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and determining the sequence by, for example the Sanger dideoxy chain termination method (see Sambrook et al).
  • Such a method generally comprises elongating, in the presence of suitable reagents, the primer by synthesis of a strand complementary to the target DNA or RNA and selectively terminating the elongation reaction at one or more of an A, C, G or T/U residue; allowing strand elongation and termination reaction to occur; separating out according to size the elongated products to determine the sequence of the nucleotides at which selective termination has occu ⁇ ed.
  • Suitable reagents include a DNA polymerase enzyme, the deoxynucleotides dATP, dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used for selective termination.
  • control regions include promoters, enhancers and locus control regions.
  • a control region we mean a nucleic acid sequence or structure which is capable of modulating the expression of a coding sequence which is operatively linked to it.
  • control regions are useful in generating transgenic animals expressing Sprouty2.
  • control regions may be used to generate expression constructs for Sprouty2. This is described in further detail below.
  • control regions of Sprouty2 are straightforward, and may be carried out in a number of ways.
  • the coding sequence of Sprouty2 may be obtained from an organism, by screening a cDNA library using a human or mouse Sprouty2 cDNA sequence as a probe.
  • 5' sequences may be obtained by screening an appropriate genomic library, or by primer extension as known in the art. Database searching of genome databases may also be employed.
  • Such 5' sequences which are particularly of interest include non-coding regions.
  • the 5' regions may be examined by eye, or with the aid of computer programs, to identify sequence motifs which indicate the presence of promoter and/or enhancer regions.
  • sequence alignments may be conducted of Sprouty2 nucleic acid sequences from two or more organisms. By aligning Sprouty2 sequences from different species, it is possible to determine which regions of the amino acid sequence are conserved between different species. Such conserved regions are likely to contain control regions for the gene in question (i.e., Sprouty2).
  • the mouse and human genomic sequences as disclosed here, for example, a mouse Sprouty2 genomic sequence, may be employed for this purpose.
  • Sprouty2 homologues from other organisms may be obtained using standard methods of screening using appropriate probes generated from the mouse and human Sprouty2 sequences.
  • the genome of the pufferfish (Takifugu rubripes) or zebrafish may also be screened to identify a S ⁇ routy2 homologue; thus, several zebrafish sequences of Sprouty2 have been identified (noted above). Comparison of the 5' non-coding region of the Fugu or zebrafish Sprouty2 gene with a mouse or human genomic Sprouty2 sequence may be used to identify conserved regions containing control regions.
  • Deletion studies may also be conducted to identify promoter and/or enhancer regions for Sprouty2.
  • putative control regions may be confirmed by molecular biology experiments, in which the candidate sequences are linked to a reporter gene and the expression of the reporter detected.
  • S ⁇ routy2 polynucleotides for example those described here, can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • we provide a method of making polynucleotides by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
  • a polynucleotide in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • operably linked means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • Vectors may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein. Vectors will be chosen that are compatible with the host cell used.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.
  • Control sequences operably linked to sequences encoding the Sprouty2 polypeptide include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in.
  • the term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters which are functional in mammalian cells, although prokaryotic promoters and promoters functional in other eukaryotic cells, such as insect cells, may be used.
  • the promoter is typically derived from promoter sequences of viral or eukaryotic genes.
  • it may be a promoter derived from the genome of a cell in which expression is to occur.
  • eukaryotic promoters they may be promoters that function in a ubiquitous manner (such as promoters of ⁇ -actin, ⁇ -actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors.
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV rous sarcoma virus
  • CMV human cytomegalovirus
  • the promoters may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • Polynucleotides may also be inserted into the vectors described above in an antisense orientation to provide for the production of antisense RNA.
  • Antisense RNA or other antisense polynucleotides may also be produced by synthetic means.
  • Such antisense polynucleotides may be used in a method of controlling the levels of RNAs transcribed from genes comprising any one of the polynucleotides described here.
  • Vectors and polynucleotides comprising or encoding Sprouty2 nucleic acids, fragments, homologues, variants or derivatives thereof may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the Sprouty2 polypeptides encoded by the polynucleotides.
  • the Sprouty2 polypeptides may be produced using prokaryotic cells as host cells, it is prefe ⁇ ed to use eukaryotic cells, for example yeast, insect or mammalian cells, in particular mammalian cells.
  • Vectors/polynucleotides may be introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses, direct injection of nucleic acids and biolistic transformation.
  • retroviruses such as retroviruses, herpes simplex viruses and adenoviruses
  • Host cells comprising polynucleotides may be used to express polypeptides, such as Sprouty2 polypeptides, fragments, homologues, variants or derivatives thereof.
  • Host cells may be cultured under suitable conditions which allow expression of the proteins.
  • Expression of the Sprouty2 polypeptides may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • Sprouty2 polypeptides can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.
  • Sprouty2 polypeptides may also be produced recombinantly in an in vitro cell-free system, such as the TnTTM (Promega) rabbit reticulocyte system.
  • TnTTM Promega rabbit reticulocyte system.
  • breast cancer There are several types of breast cancer. The most common is ductal carcinoma, which begins in the lining of the milk ducts of the breast. Another type, lobular carcinoma, begins in the lobules where breast milk is produced. If a malignant tumor invades nearby tissue, it is known as infiltrating or invasive cancer. When breast cancer spreads outside the breast, cancer cells often are found in the lymph nodes under the arm. Breast cancer cells may spread beyond the breast such as to other lymph nodes, the bones, liver, or lungs.
  • the recognised stages of breast cancer comprise:
  • Stage 0 Very early breast cancer. This type of cancer has not spread within or outside the breast. It is sometimes called DCIS, LCIS, or breast cancer in situ or non- invasive cancer.
  • Stage I The cancer is no larger than about 1 inch in size and has not spread outside the breast, (also described as early breast cancer.)
  • Stage II The presence of any of the following: the cancer is no larger than 1 inch, but has spread to the lymph nodes under the arm; the cancer is between 1 and 2 inches. It may or may not have spread to the lymph nodes under the arm; the cancer is larger than 2 inches, but has not spread to the lymph nodes under the arm.
  • Stage III and Stage IIIA The presence of any of the following: the cancer is smaller than 2 inches and has spread to the lymph nodes under the arm, the cancer also is spreading further to other lymph nodes; the cancer is larger than 2 inches and has spread to the lymph nodes under the arm.
  • Stage IIIB The presence of any of the following: the cancer has spread to tissues near the breast (skin, chest wall, including the ribs and the muscles in the chest); the cancer has spread to lymph nodes inside the chest wall along the breast bone.
  • Stage rV The cancer has spread to other parts of the body, most often the bones, lungs, liver, or brain. Or, the tumor has spread locally to the skin and lymph nodes inside the neck, near the collarbone.
  • Inflammatory breast cancer is a rare, but very serious, aggressive type of breast cancer.
  • the breast may look red and feel warm. There may be ridges, welts, or hives on the breast; or the skin may look wrinkled. It is sometimes misdiagnosed as a simple infection.
  • Recu ⁇ ent disease means that the cancer has come back (recu ⁇ ed) after it has been treated. It may come back in the breast, in the soft tissues of the chest (the chest wall), or in another part of the body.
  • breast cancer in situ Many breast cancers being found are very early cancers known as breast cancer in situ or noninvasive cancer. Most of these cancers are found by mammography. These very early cell changes may become invasive breast cancer. Two types of breast cancer in situ include the following:
  • DCIS ductal carcinoma in situ
  • ductal carcinoma in situ which means that abnormal cells are found only in the lining of a milk duct of the breast. The abnormal cells have not spread outside the duct. They have not spread within the breast, beyond the breast, to the lymph nodes under the arm, or to other parts of the body.
  • DCIS ductal carcinoma in situ
  • LCIS lobular carcinoma in situ
  • LCIS lobular carcinoma in situ
  • a biopsy is done for another lump or unusual change that is found on a mammogram.
  • Patients with LCIS have a 25 percent chance of developing breast cancer in either breast during the next 25 years.
  • Microcalcifications are very small specks of calcium that can't be felt, but can be seen on a mammogram. They are formed by rapidly dividing cells. When they are clustered in one area of the breast, this could be an early sign of breast cancer in situ. About half of the breast cancers found by mammography appear as clusters of microcalcifications. The other half appear as lumps.
  • Our diagnostic methods may be used in conjunction with any known method of diagnosis of breast cancer, including detecting of mutations in either or both of the known breast cancer genes BRCA1 and BRCA2.
  • the diagnosis may be ca ⁇ ied out by detection of Her2 expression, for example by use of anti-Her2 antibody.
  • Known treatments for breast cancer may consist of any one or more of the following: Surgery, radiation therapy, chemotherapy, high-dose chemotherapy, hormonal therapy and immunotherapy. Accordingly, any of the treatment methods described here may be combined with any one or more of the preceding known therapies. In addition, any one or more of the following general therapies known to be effective for treatment or alleviation of cancer may be used.
  • Nonspecific immunomodulating agents are substances that stimulate or indirectly augment the immune system. Often, these agents target key immune system cells and cause secondary responses such as increased production of cytokines and immunoglobulins.
  • Two nonspecific immunomodulating agents used in cancer treatment are bacillus Calmette-Guerin (BCG) and levamisole.
  • BRMs biological response modifiers
  • IFN Interferons
  • interferon alpha is the type most widely used in cancer treatment.
  • Interferons can improve the way a cancer patient's immune system acts against cancer cells.
  • mterferons may act directly on cancer cells by slowing their growth or promoting their development into cells with more normal behavior.
  • Some interferons may also stimulate NK cells, T cells, and macrophages, boosting the immune system's anticancer function.
  • interleukins are cytokines that occur naturally in the body. Many interleukins have been identified; interleukin-2 (IL-2 or aldesleukin) has been the most widely studied in cancer treatment. IL-2 stimulates the growth and activity of many immune cells, such as lymphocytes, that can destroy cancer cells.
  • IL-2 interleukin-2
  • aldesleukin aldesleukin
  • CSFs Colony-Stimulating Factors
  • Colony-stimulating factors (sometimes called hematopoietic growth factors) usually do not directly affect tumor cells; rather, they encourage bone ma ⁇ ow stem cells to divide and develop into white blood cells, platelets, and red blood cells. Bone ma ⁇ ow is critical to the body's immune system because it is the source of all blood cells.
  • G-CSF filgrastim
  • GM-CSF sertimostim
  • G- CSF and GM-CSF can increase the number of white blood cells, thereby reducing the risk of infection in patients receiving chemotherapy.
  • G- CSF and GM-CSF can also stimulate the production of stem cells in preparation for stem cell or bone ma ⁇ ow transplants; Erythropoietin can increase the number of red blood cells and reduce the need for red blood cell transfusions in patients receiving chemotherapy: and Oprelvekin can reduce the need for platelet transfusions in patients receiving chemotherapy.
  • MOABs Monoclonal Antibodies
  • Herceptin is used to treat metastatic breast cancer in patients with tumors that produce excess amounts of a protein called HER-2. (Approximately 25 percent of breast cancer tumors produce excess amounts of HER-2).
  • the methods of treatment described here may be used in combination with administration of anti-Her2 antibody, for example, Herceptin, to the individual concerned.
  • the HER-2/neu (erbB-2) gene product is a 185-kDA transmembrane receptor tyrosine kinase that belongs to the family of receptors for epidermal growth factor. It is described in some detail in Reese, D. M., et al., Stem Cells, 15, 1-8 (1997) which is incorporated herein by reference.
  • HER-2/neu is overexpressed in 20-30% of human breast cancers and the increased expression has been associated with poor prognosis.
  • the discovery of this has led to the development of HERCEPTIN, an antibody to HER-2/neu, which in tests has been found to lengthen remission time in metastatic breast cancer.
  • HER-2/neu is a cell- surface receptor that transmits growth signals to the cell nucleus. HERCEPTIN appears to block these signals thereby apparently inhibiting proliferation of cells mediated by HER- 2/neu in HER-2/neu positive breast cancer.
  • Overexpression of HER-2/neu has also been found in a portion of ovarian cancers, gastric cancers, endometrial cancers, salivary cancers, pancreatic cancers, prostate cancers, colorectal cancers, and non-small-cell lung cancers.
  • the other cancers associated with overexpression of HER-2-neu are potentially treatable with HERCEPTIN.
  • our methods of diagnosis may be combined with detection of over- expression of Her2 in an individual.
  • the methods of treatment described here may include administration of Herceptin to an individual, in addition to increasing expression of hSprouty2.
  • the anti-Her2 antibody comprises Herceptin.
  • a method of diagnosis of breast cancer comprising detecting modulation of expression of Sprouty2, preferably down-regulation of expression of Sprouty2 in a cell or tissue of an individual.
  • the presence and quantity of Sprouty2 polypeptides and nucleic acids may be detected in a sample.
  • the Sprouty2 associated diseases can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level, preferably a decreased level, of the Sprouty2 polypeptide or Sprouty2 mRNA.
  • the sample may comprise a cell or tissue sample from an organism or individual suffering or suspected to be suffering from a disease associated with increased, reduced or otherwise abnormal Sprouty2 expression, including spatial or temporal changes in level or pattern of expression.
  • the level or pattern of expression of Sprouty2 in an organism suffering from or suspected to be suffering from such a disease may be usefully compared with the level or pattern of expression in a normal organism as a means of diagnosis of disease.
  • the sample preferably comprises a cell or tissue sample from an individual suffering or suspected to be suffering from breast cancer, preferably a breast tissue or cell sample.
  • a decreased level of expression of Sprouty2 is detected in the sample.
  • the level of Sprouty2 is decreased to a significant extent when compared to normal cells, or cells known not to be cancerous. Such cells may be obtained from the individual being tested, or another individual, preferably matched to the tested individual by age, weight, lifestyle, etc.
  • the level of expression of Sprouty2 is reduced by 10%, 20%o, 30%) or 40%) or more. In highly prefe ⁇ ed embodiments, the level of expression of Sprouty2 is reduced by 45% or more, preferably 50% or more, as judged by cDNA hybridisation. As described in the Examples, when a Sprouty2 probe is hybridized to the cDNA of 50 matched normal and tumour patients, there is a reduction of more than 50% of the radioactive signal from the breast tumour cDNA when compared to normal breast cDNA. This reduction in signal was observed in 96% of the patients (patient sample size is 50).
  • Sprouty2 may be detected in a number of ways, as known in the art. Typically, the amount of Sprouty2 in a sample of tissue from an individual is measured, and compared with a sample from an unaffected individual. Both Sprouty2 nucleic acid, as well as Sprouty2 polypeptide levels may be measured.
  • nucleic acid probe which is specific for the S ⁇ routy2 nucleic acid and monitoring said sample for the presence of the Sprouty2 nucleic acid.
  • the nucleic acid probe may specifically bind to the Sprouty2 nucleic acid, or a portion of it, and bmding between the two detected; the presence of the complex itself may also be detected.
  • Sprouty2 mRNA may be measured in a sample.
  • Sprouty2 mRNA may be assayed by in situ hybridization, Northern blotting and reverse transcriptase— polymerase chain reaction.
  • Nucleic acid sequences may be identified by in situ hybridization, Southern blotting, single strand conformational polymorphism, PCR amplification and DNA-chip analysis using specific primers.
  • Sprouty2 a probe from a Sprouty2 sequence, for example, any portion of a suitable human Sprouty2 sequence may be used as a probe.
  • Prefe ⁇ ed sequences for designing Sprouty2 probes include a sequence having accession number NM_005842" or a sequence having accession number AF039843.
  • polymerase chain reaction may be employed to detect S ⁇ routy2 mRNA.
  • PCR polymerase chain reaction
  • the procedure basically involves: (1) treating extracted DNA to form single- stranded complementary strands; (2) adding a pair of oligonucleotide primers, wherem one primer of the pair is substantially complementary to part of the sequence in the sense strand and the other primer of each pair is substantially complementary to a different part of the same sequence in the complementary antisense strand; (3) annealing the paired primers to the complementary sequence; (4) simultaneously extending the annealed primers from a 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer wherein said extension products after separation from the complement serve as templates for the synthesis of an extension product for the other primer of each pair; (5) separating said extension products from said templates to produce single-stranded molecules; and (6) amp
  • RT-PCR reverse transcription-polymerase chain reaction
  • Quantitative RT-PCR is particularly prefe ⁇ ed.
  • Such PCR techniques are well known in the art, and may employ any suitable primer from a S ⁇ routy2 sequence.
  • Sprouty2 expression may be detected by detecting the presence or amount of S ⁇ routy2 polypeptide in a sample.
  • a method of detecting the presence of a Sprouty2 polypeptide by contacting a cell sample with an antibody capable of binding the polypeptide and monitoring said sample for the presence of the polypeptide. This may conveniently be achieved by monitoring the presence of a complex formed between the antibody and the polypeptide, or monitoring the bmding between the polypeptide and the antibody.
  • Methods of detecting binding between two entities are known in the art, and include FRET (fluorescence resonance energy transfer), surface plasmon resonance, etc.
  • the Sprouty2 polypeptide is detected using an anti- Sprouty2 antibody.
  • anti-Sprouty2 antibody Such antibodies are known in the art, and may for example be obtained from commercial companies.
  • An anti-human Sprouty2 antibody is sold by Abeam (catalog number abl043), and comprises a rabbit polyclonal to His tagged full length human Sprouty 2.
  • an anti-human Sprouty2 antibody may be obtained from Upstate (catalog number 07-152); this comprises a rabbit polyclonal antibody to residues 1-207 of human Sprouty 2.
  • Sprouty2 in a sample derived from a host are well-known to those of skill in the art.
  • the specimen may be assayed for polypeptides/proteins by immunohistochemical and immunocytochemical staining (see generally Stites and Ten, Basic and Clinical Immunology, Appleton and Lange, 1994), ELISA, RLA, immunoblots, Western blotting, immunoprecipitation, functional assays and protein truncation test.
  • Other assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies may be used in the assays.
  • immunoassays such as radioimmunoassays (RIA)
  • RIA radioimmunoassays
  • the diagnostic kit comprises means for detecting expression of Sprouty2 in the individual, by any means as described in this document.
  • the diagnostic kit may therefore comprise any one or more of the following: a Sprouty2 polynucleotide or a fragment thereof; a complementary nucleotide sequence to Sprouty2 nucleic acid or a fragment thereof; a Sprouty2 polypeptide or a fragment thereof, or an antibody to a Sprouty2, preferably comprising E an anti-human Sprouty2 antibody is sold by Abeam (catalogue number abl043), which comprises a rabbit polyclonal to His tagged full length human Sprouty 2, as well as an anti-human Sprouty2 antibody from Upstate (catalogue number 07-152) comprising a rabbit polyclonal antibody to residues 1- 207 of human Sprouty2.
  • the diagnostic kit may comprise instructions for use, or other indicia.
  • the diagnostic kit may further comprise means for treatment or prophylaxis of breast cancer, such as any of the compositions described in this document, or any means known in the art for treating breast cancer.
  • the diagnostic kit may comprise a therapeutic drug such as Tamoxifen (Nolvadex) or its variants such as tamoxifen tamoxifen citrate or any other antiestrogen or estrogen blocker.
  • the therapeutic drug may also comprise an anti- Sprouty2 antibody.
  • Breast cancer may be treated or prevented by administration of Sprouty2 in whole or in part, whether in the form of a Sprouty2 polypeptide, or a nucleic acid encoding Sprouty2. Treatment may also be effected by administration of a molecule identified as being capable of up-regulating the activity or expression of Sprouty2 to an individual. Such a compound may be administered along with a pharmaceutically acceptable carrier in an amount effective to activate expression or activity Sprouty2, or by activating a second signal, and thereby alleviating the abnormal condition.
  • gene therapy may be employed to effect the endogenous production of Sprouty2 by the relevant cells such as breast cells in the subject.
  • a polynucleotide encoding Sprouty2 or a portion of this may be engineered for expression in a replication defective refroviral vector, as discussed above.
  • the refroviral expression construct may then be isolated and introduced into a packaging cell transduced with a refroviral plasmid vector containing RNA encoding a S ⁇ routy2 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 S ⁇ routy2 polypeptide in vivo.
  • gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).
  • the level of Sprouty2 is increased in a breast cell. Furthermore, in such prefe ⁇ ed embodiments, treatment is targeted to, or specific to, breast cells.
  • the expression of Sprouty2 is preferably specifically increased only in diseased breast cells (i.e., those cells which are cancerous), and not substantially in other non-diseased breast cells. In the prefe ⁇ ed methods, expression of Sprouty2 is not substantially elevated in other cells, i.e., cells which are not breast cells. Thus, in such embodiments, the level of Sprouty2 remains substantially the same or similar in non-breast cells in the course of or following treatment.
  • Breast cell specific elevation of Sprouty2 levels may be achieved by targeted administration, i.e., applying Sprouty2 polypeptide or nucleic acid only to the breast cells and not other cells.
  • up-regulation of Sprouty2 expression in breast cells is employed. Such methods may advantageously make use of breast specific expression vectors, as described in further detail below.
  • Cancer gene therapy has to selectively target tumour tissues so as to reduce undesired side effects in normal tissue.
  • Targeting transgene expression to malignant tissues requires the use of specific regulatory elements including promoters based on tumour biology, tissue-specific promoters and inducible regulatory elements (Al). Promoters based on Tumour Biology
  • genes are upregulated in breast cancer.
  • the promoters of these genes can be used to drive tumour-selective expression of a transgene using a recombinant replication-defective refroviral vectors.
  • examples of such genes include the vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-1 (VEGFR- 1) and VEGFR-2, which are known to be upregulated in breast cancer in a tumour-stage dependent manner (A2).
  • VEGF vascular endothelial growth factor
  • VEGFR-2 vascular endothelial growth factor receptor-1
  • c-erbB2 oncogene is selectively upregulated in jbreast carcinomas (A3, A6).
  • L-plastin a human actin-bindng protein is constitutively and abundantly expressed in malignant epithelial cells but not in normal tissue, except for low-level expression in mature hematopoietic cells (A4).
  • Anti-apoptotic gene Bcl-2 has been found to be upregulated in breast cancer cells (A5).
  • Human breast tumours express high levels of MUCl compared to normal breast tissues (A7).
  • genes are expressed specifically in breast tissues. Examples of such genes are the human ⁇ -lactalbumin (ALA) and ovine ⁇ -lactoglobulin (BLG).
  • the promoters of such genes can be used to drive the expression of fransgenes in adenoviral vectors in a breast cancer cell-specific manner (A8).
  • Gene therapy for breast carcinoma may be approached by tailoring a virus with affinity to this tissue, such as the mouse mammary tumour virus (MMTV).
  • MMTV mouse mammary tumour virus
  • the glucorticoid-responsive long terminal repeats (LTR) of this retrovirus can be used as promoter for glucocorticoid-induced the expression of a transgene (A9).
  • Inducible promoters are used as mediators of transient transgene expression.
  • Various stress genes are upregulated in breast tumours upon i ⁇ adiation or chemotherapeutic freatment. Examples of such stress genes are heat shock protein (HSP) (A10) and multidrug resistance gene-1 (MDR-1) (Al 1).
  • HSP heat shock protein
  • MDR-1 multidrug resistance gene-1
  • the promoters of these genes can therefore be used to drive the tumour specific expression of a transgene in breast cancers that have been subjected to i ⁇ adiation or chemotherapy.
  • Transcriptionally targeted gene therapy is usually achieved by direct intratumour injection of a replication-defective adenoviral expression vector containing the transgene of interest (A6, A12, A13).
  • the transgene can also be delivered by infratumoural injection as a lipid complex with cationic liposomes (A14, Al 5).
  • antibodies which bind to a Sprouty2 polypeptide, fragment, homologue, variant or derivative thereof are useful in detecting Sprouty2 expression, and in particular in diagnosing a S ⁇ routy2 associated disease such as breast cancer.
  • Other prefe ⁇ ed antibodies include those which have therapeutic activity, i.e., which are may be used in a therapeutic manner to treat, manage or prevent any Sprouty2 associated disease, including breast cancer.
  • antibodies capable of bmding to Sprouty2 include an anti-human Sprouty2 antibody is sold by Abeam (catalogue number abl043), which comprises a rabbit polyclonal to His tagged full length human Sprouty 2, as well as an anti-human Sprouty2 antibody from Upstate (catalogue number 07-152) comprising a rabbit polyclonal antibody to residues 1-207 of human Sprouty2.
  • antibodies which are specific for Sprouty2 may be generated against any suitable epitope, for example, an epitope derived from the N terminus of the protein.
  • the sequence of a suitable N terminal fragment of S ⁇ routy2 is depicted as SEQ ID NO: 2, and any epitope from this sequence may be used for the generation of specific Sprouty2 antibodies.
  • the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
  • antibodies with fully human variable regions (or their fragments), for example, as described in US Patent Nos. 5,545,807 and 6,075,181 may also be used.
  • Neutralizing antibodies i.e., those which inhibit any biological activity of Sprouty2, are especially prefe ⁇ ed for diagnostics and therapeutics.
  • Antibodies may be produced by standard techniques, such as by immunisation or by using a phage display library. Such an antibody may be capable of binding specifically to the Sprouty2 protein or homologue, fragment, etc.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • an immunogenic composition comprising a Sprouty2 polypeptide or peptide.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified the substance amino acid sequence is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.
  • Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from a Sprouty2 polypeptide contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, we also provide Sprouty2 amino acid sequences or fragments thereof haptenised to another amino acid sequence for use as immunogens in animals or humans.
  • Monoclonal antibodies directed against epitopes obtainable from a S ⁇ routy2 polypeptide or peptide can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Ban virus.
  • Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
  • Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).
  • Antibodies both monoclonal and polyclonal, which are directed against epitopes obtainable from a Sprouty2 polypeptide or peptide are particularly useful in diagnosis.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti- idiotype antibodies are immunoglobulins which carry an "internal image" of the substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833- 3837), and Winter G and Milstein C (1991; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for the polypeptide or peptide may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • Anti-Sprouty2 antibodies may be used in method of detecting a Sprouty2 polypeptide present in biological samples by a method which comprises: (a) providing an anti-Sprouty2 antibody; (b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said antibody is formed.
  • Suitable samples include extracts tissues such as brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues or from neoplastic growths derived from such tissues.
  • a prefe ⁇ ed sample comprises a breast tissue, preferably a breast tissue from an individual suspected to be suffering from breast cancer.
  • Antibodies may be bound to a solid support and or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
  • the Sprouty2 proteins and nucleic acids may be employed in a screening process for compounds which bind the Sprouty2 polypeptides and which activate (agonists) its activity or inhibit activation of (antagonists) of Sprouty2. Such molecules are useful in the treatment methods described here. Screening assays may also be conducted on animals transgenic for Sprouty2, as described in further detail in the section below.
  • Sprouty2 may therefore be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991). Furthermore, screens may be conducted to identify factors which influence the expression of S ⁇ routy2, in particular in breast cells.
  • the assays for agonists and antagonists rely on determining the effect of candidate molecules on one or more activities of Sprouty2.
  • An assay may involve assaying Sprouty2 activity in the presence of a candidate molecule, and optionally in the absence of the candidate molecule, or in the presence of a molecule known to inhibit or activate a Sprouty2 activity.
  • Sprouty2 is responsible for many biological functions, as described in the references (in particular, refs 1 to 12). We have demonstrated that expression of Sprouty2 is decreased in breast cancer cells; accordingly, control of Sprouty2 expression may be employed to treat breast cancer and other cancers. Therefore, it is desirous to find compounds and drugs which stimulate the expression and/or activity of Sprouty2, or which can inhibit the function of this protein. In general, agonists and antagonists are employed for therapeutic and prophylactic purposes for any known cancer, in particular, breast cancer.
  • any of the activities of Sprouty2 as described above may be assayed in the presence of candidate molecules to determine their effects (if any) on these activities.
  • an assay for agonists and antagonists of Sprouty2 may detect the effect of a candidate molecule on any one or more of the following activities: inhibition of one or more events downstream of receptor tyrosine kinases; inhibition of activation of the Ras/Raf/MAPK pathway; inhibition of the Ras/Raf/MAPK pathway by preventing the activation of Ras/Raf; down-regulation of ERK phosphorylation when the Ras/MAPK pathway is activated.
  • Rational design of candidate compounds likely to be able to interact with S ⁇ routy2 may be based upon structural studies of the molecular shapes of a Sprouty2 polypeptide.
  • One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., X-ray crystallography or two-dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions.
  • X-ray crystallography or two-dimensional NMR techniques.
  • An alternative to rational design uses a screening procedure which involves in general producing appropriate cells which express the Sprouty2 proteins on the surface thereof.
  • Such cells include cells from animals, yeast, Drosophila or E. coli.
  • Cells expressing the Sprouty2 polypeptide (or cell membrane containing the expressed Sprouty2 polypeptide) are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • a test compound For example, Xenopus oocytes may be injected with mRNA encoding any one or more of the Sprouty2 polypeptides.
  • candidate compounds are proteins, in particular antibodies or peptides
  • libraries of candidate compounds may be screened using phage display techniques.
  • Phage display is a protocol of molecular screening which utilises recombinant bacteriophage.
  • the technology involves transforming bacteriophage with a gene that encodes one compound from the library of candidate compounds, such that each phage or phagemid expresses a particular candidate compound.
  • the transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate candidate compound and displays it on their phage coat.
  • Specific candidate compounds which are capable of binding to a Sprouty2 polypeptide or peptide are enriched by selection strategies based on affinity interaction. The successful candidate agents are then characterised.
  • Phage display has advantages over standard affinity ligand screening technologies.
  • the phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occurring conformation. This allows for more specific and higher affinity binding for screening purposes.
  • Another method of screening a library of compounds utilises eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a library of compounds. Such cells, either in viable or fixed form, can be used for standard binding-partner assays. See also Parce et al. (1989) Science 246:243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87;4007-4011, which describe sensitive methods to detect cellular responses.
  • This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic following by washing, or centrifugation of the cell membranes.
  • Still another approach is to use solubilized, unpurified or solubilized purified polypeptide or peptides, for example extracted from transformed eukaryotic or prokaryotic host cells. This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.
  • Another technique for candidate compound screening involves an approach which provides high throughput screening for new compounds having suitable binding affinity, e.g., to a Sprouty2 polypeptide, and is described in detail in International Patent application no. WO 84/03564 (Commonwealth Serum Labs.), published on September 13 1984.
  • a solid substrate e.g., plastic pins or some other appropriate surface; see Fodor et al. (1991).
  • all the pins are reacted with solubilized Sprouty2 polypeptide and washed.
  • the next step involves detecting bound polypeptide. Compounds which interact specifically with the Sprouty2 polypeptide will thus be identified.
  • Ligand binding assays provide a direct method for ascertaining pharmacology and are adaptable to a high throughput format.
  • the purified ligand for a Sprouty2 polypeptide may be radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its binding partner.
  • Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell sources. For these assays, specific binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the Sprouty2 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by binding to the Sprouty2 polypeptide, using detection systems appropriate to the cells bearing the polypeptides at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing a S ⁇ routy2 polypeptide to form a mixture, measuring activity of the relevant protein in the mixture, and comparing the activity of the mixture to a standard.
  • the assays may involve exposing a candidate molecule to a cell, preferably a breast cell, and assaying expression of Sprouty2 by any suitable means.
  • Molecules which up-regulate the expression of Sprouty2 in such assays may be optionally chosen for further study, and used as drugs to elevate Sprouty2 expression. Such drugs may be usefully employed to treat or prevent breast cancer.
  • cDNA encoding S ⁇ routy2 protein and antibodies to the proteins may also be used to configure assays for detecting the effect of added compounds on the production of mRNA and protein in cells.
  • an ELISA may be constructed for measuring secreted or cell associated levels of Sprouty2 polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of Sprouty2 protein (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well understood in the art.
  • Examples of potential antagonists of Sprouty2 include antibodies or, in some cases, nucleotides and their analogues, including purines and purine analogues, oligonucleotides or proteins which are closely related to a binding partner of Sprouty2, e.g., a fragment of the binding partner, or small molecules which bind to the Sprouty2 polypeptide but do not elicit a response, so that the activity of the polypeptide is prevented.
  • compound refers to a chemical compound (naturally occurring or synthesised), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an exfract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
  • a biological macromolecule e.g., nucleic acid, protein, non-peptide, or organic molecule
  • an exfract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
  • the compound is an antibody.
  • the materials necessary for such screening to be conducted may be packaged into a screening kit.
  • a screening kit is useful for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for Sprouty2 polypeptides or compounds which decrease or enhance the production of Sprouty2.
  • the screening kit may comprise: (a) a Sprouty2 polypeptide; (b) a recombinant cell expressing a Sprouty2 polypeptide; (c) a cell membrane expressing a Sprouty2 polypeptide; or (d) an antibody to Sprouty2 polypeptide.
  • the screening kit may optionally comprise instructions for use.
  • kits may also be provided which are capable of detecting Sprouty2 expression at the nucleic acid level.
  • Such kits may comprise a primer for amplification of Sprouty2, for example, having or comprising the sequence 5'
  • kits may comprise a nucleic acid probe for Sprouty2 expression, as described in this document.
  • the kits may also optionally comprise instructions for use.
  • transgenic animals capable of expressing natural or recombinant Sprouty2, or a homologue, variant or derivative, at elevated or reduced levels compared to the normal expression level.
  • the transgenic animals are useful as models of cancer, particularly breast cancer. Furthermore, they may be used for screening compounds and molecules capable of modulating Sprouty2 expression or function. Such molecules are useful for the methods of freatment described here.
  • the Sprouty2 knockou 's may arise as a result of functional disruption of the Sprouty2 gene or any portion of that gene, including one or more loss of function mutations, including a deletion or replacement, of the Sprouty2 gene.
  • the mutations include single point mutations, and may target coding or non-coding regions of Sprouty2.
  • such a transgenic animal is a non-human mammal, such as a pig, a sheep or a rodent. Most preferably the transgenic animal is a mouse or a rat.
  • Such transgenic animals may be used in screening procedures to identify agonists and/or antagonists of Sprouty2, as well as to test for their efficacy as treatments for diseases in vivo.
  • Mice which are null for Sprouty2 may be used for various purposes.
  • transgenic animals that have been engineered to be deficient in the production of Sprouty2 may be used in assays to identify agonists and/or antagonists of Sprouty2.
  • One assay is designed to evaluate a potential drug (a candidate ligand or compound) to determine if it produces a physiological response in the absence of Sprouty2. This may be accomplished by administering the drug to a transgenic animal as discussed above, and then assaying the animal for a particular response. Although any physiological parameter could be measured in this assay, prefe ⁇ ed responses include altered tumour susceptability, particularly, altered susceptability to developing breast cancer, altered neovascularization.
  • Tissues derived from the Sprouty2 knockout animals may be used in binding assays to determine whether the potential drug (a candidate ligand or compound) binds to Sprouty2.
  • Such assays can be conducted by obtaining a first preparation from the transgenic animal engineered to be deficient in Sprouty2 production and a second preparation from a source known to bind any identified Sprouty2 ligands or compounds.
  • the first and second preparations will be similar in all respects except for the source from which they are obtained.
  • breast tissue from a transgenic animal such as described above and below
  • comparable breast tissue from a normal (wild type) animal is used as the source of the second preparation.
  • Each of the preparations is incubated with a ligand known to bind to Sprouty2, both alone and in the presence of the candidate ligand or compound.
  • the candidate ligand or compound will be examined at several different concentrations.
  • Tissues derived from transgenic animals may be used in assays directly or the tissues may be processed to isolate extracts or proteins, which are themselves used in the assays.
  • a prefe ⁇ ed transgenic animal is the mouse.
  • the ligand may be labeled using any means compatible with binding assays. This would include, without limitation, radioactive, enzymatic, fluorescent or chemiluminescent labeling (as well as other labelling techniques as described elsewhere).
  • antagonists of Sprouty2 may be identified by administering candidate compounds, etc, to wild type animals expressing functional Sprouty2, and animals identified which exhibit any of the phenotypic characteristics associated with reduced or abolished expression of Sprouty2 function, such as breast cancer or susceptibility to breast cancer.
  • the transgenic animal is one which is more susceptible to developing cancer, preferably breast cancer, as compared to a wild type animal or an animal which expresses Sprouty2.
  • a wild type animal or an animal which expresses Sprouty2 Such an animal may be used as a model for breast cancer, to study the mechanisms which cause or are co ⁇ elated to development of breast cancer.
  • transgenic Sprouty2 animals may be used to screen for molecules which affect the development of cancer such as breast cancer in the fransgenic animal.
  • an assay may comprise exposing the fransgenic animal to a candidate molecule, and monitoring the development of breast cancer in the animal.
  • Candidate molecules which reduce or abolish development of breast cancer, or which slow down the progress of the cancer once developed, in the transgenic animal (as compared to a transgenic Sprouty2 animal which has not been exposed to the candidate molecule) may be selected as potential drugs for treatment of cancer in mammals, for example humans.
  • the assay may be modified by allowing the fransgenic animal to develop breast cancer, and then exposing the animal to a candidate molecule.
  • suitable candidate breast cancer therapies may be selected from those molecules which reduce the already developed breast cancer, for example, enable the animal to go into remission.
  • Transgenic gene constructs can be introduced into the germ line of an animal to make a transgenic mammal. For example, one or several copies of the construct may be incorporated into the genome of a mammalian embryo by standard transgenic techniques.
  • the transgenic non-human animals are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor.
  • the transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
  • the Sprouty2 transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s).
  • the egg may be incubated in vitro for varying amounts of time, or reimplanted into the su ⁇ ogate host, or both. In vitro incubation to maturity is within the scope of the methods and compositions described here.
  • the progeny of the transgenically manipulated embryos can be tested for the presence of the construct by Southern blot analysis of the segment of tissue. If one or more copies of the exogenous cloned construct remains stably integrated into the genome of such transgenic embryos, it is possible to establish permanent transgenic mammal lines carrying the transgenically added construct.
  • the litters of transgenically altered mammals can be assayed after birth for the incorporation of the construct into the genome of the offspring.
  • this assay is accomplished by hybridizing a probe co ⁇ esponding to the DNA sequence coding for the desired recombinant protein product or a segment thereof onto chromosomal material from the progeny.
  • Those mammalian progeny found to contain at least one copy of the construct in their genome are grown to maturity.
  • a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
  • the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
  • the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
  • a euploid zygote is prefe ⁇ ed. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
  • the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
  • the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. There will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences. Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
  • Reimplantation is accomplished using standard methods. Usually, the su ⁇ ogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
  • Transgenic offspring of the su ⁇ ogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the fransgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
  • DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the fransgene.
  • the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
  • Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
  • Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
  • the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
  • the partner may be a parental line.
  • in vitro fertilization is used, the fertilized embryo may be implanted into a su ⁇ ogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • the fransgenic ammals will include exogenous genetic material.
  • the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a S ⁇ routy2 polypeptide. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the fransgene product in a specific type of cell.
  • Refroviral infection can also be used to introduce transgene into a non-human animal.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for refroviral infection (Jaenich, R. (1976) PNAS 73 : 1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
  • the viral vector system used to introduce the fransgene is typically a replication-defective refrovirus carrying the transgene (Jahner et al.
  • ES embryonal stem cell
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated fransduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240:1468-1474.
  • transgenic animal is characterized by having an altered Sprouty2 gene, preferably as described above, as models for Sprouty2 function, in particular, models for breast cancer.
  • Alterations to the gene include deletions or other loss of function mutations, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations, introduction of an exogenous gene from another species, or a combination thereof.
  • the fransgenic animals may be either homozygous or heterozygous for the alteration.
  • the animals and cells derived therefrom are useful for screening biologically active agents that may modulate Sprouty2 function.
  • the screening methods are of particular use for determining the specificity and action of potential therapies for S ⁇ routy2 associated diseases including cancers such as breast cancer.
  • the animals are useful as a model to investigate the role of Sprouty2 in normal breast and other tissues.
  • Another aspect pertains to a fransgenic nonhuman animal having a functionally disrupted endogenous Sprouty2 gene but which also carries in its genome, and expresses, a transgene encoding a heterologous Sprouty2 protein (i.e., a Sprouty2 from another species).
  • the animal is a mouse and the heterologous Sprouty2 is a human Sprouty2.
  • An animal, or cell lines derived from such an animal, which has been reconstituted with human S ⁇ routy2, can be used to identify agents that inhibit human Sprouty2 in vivo and in vitro.
  • a stimulus that induces signalling through human Sprouty2 can be administered to the animal, or cell line, in the presence and absence of an agent to be tested and the response in the animal, or cell line, can be measured.
  • An agent that inhibits human Sprouty2 in vivo or in vitro can be identified based upon a decreased response in the presence of the agent compared to the response in the absence of the agent.
  • “Sprouty2 knock-out” or a “Sprouty2 null” Such an animal is one which expresses lowered or no Sprouty2 activity, preferably as a result of an endogenous Sprouty2 genomic sequence being disrupted or deleted.
  • the endogenous Sprouty2 genomic sequence may be replaced by a null allele, which may comprise non-functional portions of the wild-type Sprouty2 sequence.
  • the endogenous Sprouty2 genomic sequence may be replaced by an allele of Sprouty2 comprising a disrupting sequence which may comprise heterologous sequences, for example, reporter sequences and/or selectable markers.
  • the endogenous Sprouty2 genomic sequence in a Sprouty2 knock-out mouse is replaced by an allele of Sprouty2 in which one or more, preferably all, of the transmembrane sequences is replaced by such a disrupting sequence, preferably a lacZ sequence and a neomycin resistance sequence.
  • the genomic Sprouty2 sequence which is functionally disrupted comprises a mouse Sprouty2 genomic sequence.
  • Sprouty2 knock-outs may be generated by various means known in the art, as described in further detail below.
  • the nucleic acid construct comprises: a) a non-homologous replacement portion; b) a first homology region located upstream of the non-homologous replacement portion, the first homology region having a nucleotide sequence with substantial identity to a first Sprouty2 gene sequence; and c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second Sprouty2 gene sequence, the second Sprouty2 gene sequence having a location downstream of the first Sprouty2 gene sequence in a naturally occurring endogenous Sprouty2 gene.
  • the first and second homology regions are of sufficient length for homologous recombination between the nucleic acid construct and an endogenous Sprouty2 gene in a host cell when the nucleic acid molecule is introduced into the host cell.
  • the non-homologous replacement portion comprises an expression reporter, preferably including lacZ and a positive selection expression cassette, preferably including a neomycin phosphofransferase gene operatively linked to a regulatory element(s).
  • Another aspect pertains to recombinant vectors into which the nucleic acid construct has been incorporated.
  • Yet another aspect pertains to host cells into which the nucleic acid construct has been introduced to thereby allow homologous recombination between the nucleic acid construct and an endogenous Sprouty2 gene of the host cell, resulting in functional disruption of the endogenous Sprouty2 gene.
  • the host cell can be a mammalian cell that normally expresses Sprouty2 from the liver, brain, spleen or heart, or a pluripotent cell, such as a mouse embryonic stem cell.
  • an embryonic stem cell into which the nucleic acid construct has been introduced and homologously recombined with the endogenous Sprouty2 gene produces a fransgenic nonhuman animal having cells that are descendant from the embryonic stem cell and thus carry the Sprouty2 gene disruption in their genome.
  • Animals that carry the Sprouty2 gene disruption in their germline can then be selected and bred to produce ammals having the Sprouty2 gene disruption in all somatic and germ cells. Such mice can then be bred to homozygosity for the Sprouty2 gene disruption.
  • a Sprouty2 deficient transgenic animal may be generated as follows:
  • Murine Sprouty2 genomic clones are isolated from a mouse large insert PAC library obtained from HGMP (Hinxton, UK) using the human open reading frame cDNA sequence as a probe using standard techniques. The isolated murine Sprouty2 genomic clones are then restriction mapped in the region of the S ⁇ routy2 gene using small oligonucleotide probes and standard techniques.
  • the murine genomic locus is partially sequenced to enable the design of homologous arms to clone into the targeting vector.
  • a 5' homologous arm and a 3' homologous arm are amplified by PCR and the fragment cloned into the targeting vector.
  • Any suitable size may be chosen for the length of these arms to enable homologous recombination; for example, the 5' arm may be between 1 kb and to 2 kb, for example 1.15 kb, while the 3 ' arm may be about 4 kb in size.
  • a targeting vector is prepared where the deleted Sprouty2 sequence is replaced with non-homologous sequences composed of an endogenous gene expression reporter (an in frame fusion with lacZ) upstream of a selection cassette composed of a self promoted neomycin phosphofransferase (neo) gene in the same orientation as the Sprouty2 gene.
  • Embryonal stem cells (Evans and Kaufman, 1981) are cultured on a neomycin resistant embryonal fibroblast feeder layer grown in Dulbecco's Modified Eagles medium supplemented with 20% Fetal Calf Serum, 10% new-born calf serum, 2 mM glutamine, non-essential amino acids, 100 ⁇ M 2-mercaptoethanol and 500 u/ml leukemia inhibitory factor. Medium is changed daily and ES cells are subcultured every three days. 5x10 6 ES cells are transfected with 5 ⁇ g of linearized plasmid by elecfroporation (25 ⁇ F capacitance and 400 Volts).
  • the transfected cells are cultured for 9 days in medium containing 200 ⁇ g/ml neomycin.
  • Clones are picked into 96 well plates, replicated and expanded before being screened by PCR to identify clones in which homologous recombination had occu ⁇ ed between the endogenous Sprouty2 gene and the targeting construct. From 200 picked clones 7 targets are identified. These clones where expanded to allow replicas to be frozen and sufficient high quality DNA to be prepared for Southern blot confirmation of the targeting event using external 5' and 3' probes, all using standard procedures (Russ et al, 2000)
  • C57BL/6 female and male mice are mated and blastocysts are isolated at 3.5 days of gestation. 10-12 cells from a chosen clone are injected per blastocyst and 7-8 blastocysts are implanted in the uterus of a pseudopregnant FI female. Five chimeric pups are born of which one male is 100% agouti (indicating cells descendent from the targeted clone). This male chimera is mated with female and MF1 and 129 mice, and germline transmission is determined by the agouti coat color and by PCR genotyping respectively.
  • Sprouty2 polypeptide Sprouty2 nucleic acid, up-regulator of Sprout2 expression or activity, molecule capable of binding to Sprouty2
  • a Cancer Profiling A ⁇ ay is purchased from Clontech (Palo Alto, CA). The a ⁇ ay consists of 241 pairs of cDNA spots generated from tumour and co ⁇ esponding normal tissue samples of individual patients spotted side-by-side on a nylon membrane.
  • N-terminal hSpry2 cDNA fragment (53 lbp) is purified from PXJ40FLAG vector after restriction digestion.
  • the cDNA is labelled by random oligonucleotide priming (High Prime DNA Labeling Kit, Mannheim, Germany) according to the manufacturer's instructions.
  • the labelled probe is purified by spin-column centrifugation (Probe QuantTM G-50 Micro Columns, Amersham Pharmacia, NJ, USA). The specific activity of the probe is lxl 0 9 cpm/ ⁇ g and the concentration of probe in the final hybridization buffer is 5-10xl0 6 cpm/ml.
  • hSpry2 probes are prepared to each of the sprouty family from regions of their respective N-termini to minimize non-specific hybridization.
  • the specificity of the hSpry2 probe is affirmed by its ability to hybridize only to hSpry2 cDNA and not to cDNAs from the other human sproutys.
  • the membrane is pre-hybridized with 10ml of pre-hybridization buffer at 65°C for 30min in a hybridization bottle subjected to continuous agitation.
  • the labeled cDNA probe is mixed with 30 ⁇ g of C 0 t-1 DNA, 150 ⁇ g of sheared salmon testes DNA and 50 ⁇ l of 20xSSC. The mixture is first heated at 95-100°C for 5min, then at 68°C for 30min, and subsequently added to the remaining 5ml of pre-hybridization buffer to reconstitute the final hybridization buffer.
  • wash buffer 2xSSC, 0.5%SDS
  • ⁇ 0.2xSSC, 0.5%SDS
  • the membrane is washed three times with 200ml of Wash buffer I and once with 200ml of Wash buffer II, each at 65°C for 30min.
  • the membrane is rinsed with 200ml of 2xSSC at room temperature for 5min with continuous agitation, before exposure to X-ray film at 70°C for various lengths of time (24hr to 3 days).
  • the membrane is stripped by boiling it in 0.5%SDS solution for 5-10min. Following this, the membrane is re-probed with a human ubiquitin confrol probe (provided by the manufacturer).
  • the intensity of the signal is quantified by densitometry (Bio-Rad, CA, USA).
  • the background is measured and subtracted off the quantified signal.
  • a scatter plot graph of the adjusted intensities (absolute intensity minus background) of the tumour versus normal tissue is plotted using Microsoft Excel software.
  • hSpry2 expression is lower in breast cancer tissue compared to normal breast tissue.
  • hSpry2 has a relatively high expression in normal tissues of these two organs, and the sample sizes are comparable to the breast tumour sample. It can be seen that the expression of hSpry2 in both of these tumours when compared with normal cell expression shows either no significant change or are only moderately different in relatively few cases.
  • tumour cells The reduced level of hSpry2 expression in tumour cells is tissue specific to breast tissue.
  • the blot is next stripped and the effectiveness of stripping is verified by exposure on x-ray film overnight.
  • the stripped blot is re-analyzed with an [ ⁇ - 32 P] dCTP-labelled ubiquitin control probe to reveal the equality of cDNA loading on the blot.
  • the resultant autoradiograph is shown in Figure 2F.
  • RNA is extracted from these tissues using standard techniques and real-time quantitative PCR is carried out using suitably designed hSpry2 primers and probes to analyze the expression level of the hSpry2 transcripts in the various tissues and organs.
  • real-time PCR is an appropriate technique for analyzing the expression level of the hSpry2 transcripts in breast tissues.
  • the expression of Sprouty 2 from 50 breast tumours is analysed and compared to the baseline expression of Sprouty 2 found in normal breast tissue (obtained by analysing 15 normal breast tissues).
  • the analysis includes comparing and co ⁇ elating the expression levels of hSpry2 with the levels of expression of other genes that have been demonstrated to be altered in breast cancers.
  • hSpry2 expression is compared with the expression of EGFR, ErbB2 and Estrogen receptor, which are known to be over expressed in some breast cancers.
  • HIN-1 and maspin expression levels are analysed. HIN-1 and maspin show repressed expression in a similar manner to hSpry2 but neither is breast specific
  • HIN-1 shows most similarity to the expression pattern of hSpry2. It is therefore advantageous to compare HIN-1 and hSpry2 expression in a side-by-side analysis.
  • HIN-1 a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells. Proc Natl Acad Sci U S A. 2001 98(17):9796-801.
  • VEGF vascular endothelial growth factor

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Abstract

La présente invention concerne une méthode de diagnostic d'un cancer chez un individu, la méthode consistant à détecter une modulation de l'expression d'une séquence de Sprouty2. L'invention concerne également une méthode de traitement ou de prophylaxie d'un cancer chez un individu, la méthode consistant à moduler la quantité d'un polypeptide ou d'un acide nucléique de Sprouty2 dans une cellule d'un individu. Lesdites méthodes sont appropriées pour la détection et le traitement du cancer du sein et sont fondées sur la découverte que l'expression de Sprouty2 est régulée négativement dans ladite maladie.
PCT/SG2003/000228 2002-09-25 2003-09-23 Methode de diagnostic du cancer Ceased WO2004029295A1 (fr)

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AU2003263728A AU2003263728A1 (en) 2002-09-25 2003-09-23 Method for diagnosis of cancer

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GB0222276.8 2002-09-25
GBGB0222276.8A GB0222276D0 (en) 2002-09-25 2002-09-25 Methods

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AU (1) AU2003263728A1 (fr)
GB (1) GB0222276D0 (fr)
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WO2006025801A3 (fr) * 2004-09-01 2006-06-08 Agency Science Tech & Res Procedes
EP2687609A3 (fr) * 2008-11-10 2014-04-23 The United States of America As Represented by the Secretary Department of Health and Human Services Méthode de traitement de tumeurs solides

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WO1998020032A1 (fr) * 1996-11-07 1998-05-14 The Board Of Trustees Of The Leland Stanford Junior University Proteine ramifiee et sequence codante
WO2001042425A2 (fr) * 1999-12-07 2001-06-14 Childrens Hospital Los Angeles Research Institute Cellules embryonnaires pulmonaires et regeneration pulmonaire
WO2001097850A2 (fr) * 2000-06-23 2001-12-27 Schering Aktiengesellschaft Combinaisons et compositions perturbant la fonction des recepteurs vegf/vegf et angiopoietine/tie et leur utilisation (ii)
WO2002059271A2 (fr) * 2001-01-25 2002-08-01 Gene Logic, Inc. Profils d'expression genetique dans des tissus mammaires
WO2003074007A2 (fr) * 2002-03-01 2003-09-12 Rigel Pharmaceuticals, Inc. Modulateurs d'activation des leucocytes, compositions, et methodes d'utilisation

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006025801A3 (fr) * 2004-09-01 2006-06-08 Agency Science Tech & Res Procedes
GB2432908A (en) * 2004-09-01 2007-06-06 Agency Science Tech & Res Methods
EP2687609A3 (fr) * 2008-11-10 2014-04-23 The United States of America As Represented by the Secretary Department of Health and Human Services Méthode de traitement de tumeurs solides
US8735082B2 (en) 2008-11-10 2014-05-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Gene signature for predicting prognosis of patients with solid tumors
US9394358B2 (en) 2008-11-10 2016-07-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Gene signature for predicting prognosis of patients with solid tumors

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GB0222276D0 (en) 2002-10-30

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