Nothing Special   »   [go: up one dir, main page]

WO2011024433A1 - Lung and esophageal cancer related gene adamts18 - Google Patents

Lung and esophageal cancer related gene adamts18 Download PDF

Info

Publication number
WO2011024433A1
WO2011024433A1 PCT/JP2010/005185 JP2010005185W WO2011024433A1 WO 2011024433 A1 WO2011024433 A1 WO 2011024433A1 JP 2010005185 W JP2010005185 W JP 2010005185W WO 2011024433 A1 WO2011024433 A1 WO 2011024433A1
Authority
WO
WIPO (PCT)
Prior art keywords
adamts18
gene
cancer
double
polypeptide
Prior art date
Application number
PCT/JP2010/005185
Other languages
French (fr)
Inventor
Yataro Daigo
Takuya Tsunoda
Yusuke Nakamura
Original Assignee
Oncotherapy Science, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oncotherapy Science, Inc. filed Critical Oncotherapy Science, Inc.
Priority to EP10811492A priority Critical patent/EP2470651A1/en
Priority to JP2012510050A priority patent/JP2013502203A/en
Priority to CN2010800480314A priority patent/CN102597235A/en
Publication of WO2011024433A1 publication Critical patent/WO2011024433A1/en

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods for detecting and diagnosing cancer as well as methods for treating and preventing cancer.
  • NPL 1 Primary lung cancer is the leading cause of cancer deaths in most countries (NPL 1), and esophageal squamous cell carcinoma (ESCC) is one of the most common fatal malignancies of the digestive tract (NPL 2).
  • ESCC esophageal squamous cell carcinoma
  • NPL 2 esophageal squamous cell carcinoma
  • NPL 3 fatal disease progression
  • NPL 4 therapeutic monoclonal antibodies and small-molecule agents
  • a number of targeted therapies such as bevacizumab, cetuximab, erlotinib, gefitinib, sorafenib, and sunitinib have been investigated in phase II and phase III trials for the treatment of advanced non-small-cell lung cancer (NSCLC; NPLs 4-8).
  • NPLs 4-5 proangiogenic protein vascular endothelial growth factor
  • cetuximab epidermal growth factor receptor
  • erlotinib and gefitinib Two small-molecule epidermal growth factor receptor tyrosine kinase inhibitors, erlotinib and gefitinib, have been shown to be effective for a subset of advanced NSCLC patients (NPLs 6-7).
  • sorafenib inhibitor for c-RAF, b-RAF, vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor h, and KIT
  • sunitinib inhibitor for platelet-derived growth factor receptor, KIT, FLT3, and vascular endothelial growth factor receptor
  • NPL 4-8) a genome-wide analysis of gene expression profiles of cancer cells from 101 lung-cancer and 19 ESCC patients was previously performed by means of a cDNA microarray consisting of 27,648 genes or expressed sequence tags (NPLs 3, 9-14).
  • NPLs 15-33 a screening system by a combination of the tumor tissue microarray analysis of clinical lung and esophageal cancer materials with RNA interference technique.
  • ADAM small cell lung cancer cells
  • ADAMTS18 an agent up-regulated in small cell lung cancer cells
  • ADAMTS members There are two subgroups in the ADAM family: membrane-anchored ADAM and secreted-type ADAMTS.
  • ADAM members are composed of common domains including propeptide, metalloproteinase, disintegrin, cysteine-rich, EGF-like, transmembrane and cytoplasmic domains, whereas ADAMTS members contain thrombospondin motifs, cysteine-rich and spacer domains in addition to propeptide, metalloproteinase and disintegrin domains (NPL 34).
  • ADAMTSs are soluble proteins, many of them appear to bind the extracellular matrix through their thrombospondin motifs or their spacer region (NPL 35). With the exception of ADAMTS10 and -12, ADAMTSs are regulated through a proteolytic processing occurring at the furin-like recognition site located between the pro- and catalytic domains (NPLs 35-36). Dysregulation of the production of several ADAMs and ADAMTSs has been documented in lung cancers (NPLs 16, 37-45) and other cancers (NPL 37), such as brain tumors, prostate cancer, liver carcinoma, breast cancer, etc. In spite of these reports, the significance of activation of ADAMTS18 in human cancer progression and its clinical potential as a therapeutic target have not yet been fully described.
  • NPL 1 Ahmedin J, Rebecca S, Elizabeth W, et al. Cancer statistics, 2007.CA Cancer J Clin 2007;57:43-66
  • NPL 2 Shimada H, Nabeya Y, Ochiai T, et al. Surgery 2003;133:486-94
  • NPL 3 Daigo Y, Nakamura Y. Gen Thorac Cardiovasc Surg 2008;56:43-53
  • NPL 4 Thatcher N. Lung Cancer 2007;57 Suppl 2:S18-23
  • NPL 5 Sandler A, Gray R, Perry MC, et al. N Engl J Med 2006;355:2542-50
  • NPL 6 Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al.
  • NPL 12 Kikuchi T, Daigo Y, Ishikawa N, et al. Int J Oncol 2006; 28:799-805
  • NPL 13 Taniwaki M, Daigo Y, Ishikawa N, et al. Int J Oncol 2006;29:567-75
  • NPL 14 Yamabuki T, Daigo Y, Kato T, et al. Int J Oncol 2006;28:1375-84
  • NPL 15 Suzuki C, Daigo Y, Kikuchi T, et al. Cancer Res 2003;63:7038-41
  • NPL 16 Ishikawa N, Daigo Y, Yasui W, et al.
  • NPL 42 Mochizuki S, Shimoda M, Shiomi T, et al. Biochem Biophys Res Commun 2004;315:79-84
  • NPL 43 Ohtsuka T, Shiomi T, Shimoda M, et al. Int J Cancer 2006;118:263-73.
  • NPL 44 Schutz A, Hartig W, Wobus M, et al. Virchows Arch 2005;446:421-9
  • NPL 45 Dunn JR, Panutsopulos D, Shaw MW, et al. Br J Cancer 2004;91:1149-54
  • the present invention relates to the discovery of a specific expression pattern of the ADAMTS18 gene in cancerous cells.
  • the ADAMTS18 gene was revealed to be frequently up-regulated in human tumors, in particular, lung and esophageal tumors.
  • small interfering RNA siRNA
  • this gene may serve as a novel therapeutic target for human lung and esophageal cancers.
  • the ADAMTS18 gene identified herein, as well as its transcription and translation products finds diagnostic utility as a marker for lung and esophageal cancer and as an oncogene target, the expression and/or activity of which may be altered to treat or alleviate a symptom of cancer.
  • various agents for treating or preventing cancer can be identified. Accordingly, it is an object of the present invention to provide a method for diagnosing or determining a predisposition to lung or esophageal cancer in a subject by determining the expression level of the ADAMTS18 gene in a subject-derived biological sample, such as tissue sample. An increase in the level of expression of the gene as compared to a normal control level indicates that the subject suffers from or is at risk of developing lung or esophageal cancer.
  • Test substances that bind the ADAMTS18 protein may be used to reduce symptoms of lung or esophageal cancer, or treating and/or preventing lung cancer or esophageal cancer.
  • the biological activity of the ADAMTS18 protein to be detected is preferably cell proliferative activity (cell proliferation enhancing activity), N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity.
  • MMP Matrix Metalloproteinase
  • the test cell may be an epithelial cell, such as cancerous epithelial cell.
  • a decrease in the expression level of the gene as compared to a control level in the absence of the test substance indicates that the test substance may be used to reduce symptoms of lung and esophageal cancer or treating and/or preventing lung cancer or esophageal cancer.
  • the present invention confirms the inhibitory effect of siRNAs for the ADAMTS18 gene.
  • the inhibition of cell proliferation of cancer cells by the siRNAs is demonstrated in the Examples section.
  • the data herein support the utility of the ADAMTS18 gene as a preferred therapeutic target for lung and esophageal cancer and the utility of double-stranded molecules against the ADAMTS18 gene as cancer therapeutics.
  • it is another object of the present invention is to provide a double-stranded molecule that inhibits the expression of the ADAMTS18 gene as well as siRNAs against the ADAMTS18 gene, and a vector encoding the double-stranded molecule.
  • a double-stranded molecule of the present invention inhibits expression of the gene when introduced into a cell expressing an ADAMTS18 gene and is composed of a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 and 12 as a target sequence, and the antisense strand has a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  • It is yet another object of the present invention is to provide a method for treating and/or preventing cancer in a subject, or inhibiting cancer cell growth.
  • Therapeutic methods of the present invention include the step of administering an antisense composition to the subject.
  • the antisense composition reduces the expression of the ADAMTS18 gene.
  • the antisense compositions may contain a nucleotide that is complementary to the ADAMTS18 gene sequence.
  • the present methods may include the step of administering an siRNA or double-stranded molecule composition to the subject.
  • the siRNA or double-stranded molecule composition reduces the expression of the ADAMTS18 gene.
  • the treatment and/or prevention of lung and esophageal cancer in a subject may be carried out by administering a ribozyme composition to the subject.
  • a ribozyme composition reduces the expression of the ADAMTS18 gene.
  • a pharmaceutical composition of the present invention includes an antisense nucleotide or double-stranded molecule (e.g., siRNA) against ADAMTS18 gene that inhibits the expression of the ADAMTS18 gene. It is yet another object of the present invention is to provide for the use of an antisense nucleotide or double-stranded molecule (e.g., siRNA) against ADAMTS18 gene, particularly in the context of cancer therapy and prevention, more particularly the treatment and/or prevention of lung and esophageal cancer.
  • siRNA antisense nucleotide or double-stranded molecule
  • the present invention provides the following [1] to [26]: [1] A method for detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of an ADAMTS18 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates that said subject suffers from or is at a risk of developing cancer, wherein said expression level is determined by any method selected from a group consisting of: (a) detecting mRNA of an ADAMTS18 gene; (b) detecting a protein encoded by an ADAMTS18 gene; and (c) detecting a biological activity of a protein encoded by an ADAMTS18 gene, [2] The method of [1], wherein said expression level is at least 10% greater than the normal control level, [3] The method of [1] or [2], wherein the biological activity is cell proliferative activity, N-glycosylation activity, invasive activity or Matrix
  • Fig. 1 depicts the expression of ADAMTS18 in lung and esophageal cancers and normal tissues: Part A depicts the expression of ADAMTS18 gene in 15 clinical lung cancers (lung ADC, lung SCC, and SCLC; top panels) and 15 lung-cancer cell lines (bottom panels), detected by semiquantitative RT-PCR analysis. Part B depicts the expression of ADAMTS18 gene in 10 clinical ESCCs (top panels), and 10 esophageal cancer cell lines (bottom panels), detected by semiquantitative RT-PCR analysis.
  • Fig. 1 depicts the expression of ADAMTS18 in lung and esophageal cancers and normal tissues: Part C depicts the expression of ADAMTS18 gene in cancerous tissues and normal tissues of 5 clinical ESCCs, detected by semiquantitative RT-PCR analysis. Part D depicts the expression of ADAMTS18 in normal human tissues, detected by Northern-blot analysis.
  • Fig. 2 depicts the inhibition of growth of NSCLC and ESCC cells by siRNAs against ADAMTS18:
  • the top panels depict the results of semiquantitative RT-PCR, confirming the expression of ADAMTS18 in response to siRNAs for ADAMTS18 (si-ADAMTS18-#1 or #2) or control siRNAs (EGFP or LUC) in DMS114 and TE4 cells, analyzed by semiquantitative RT-PCR.
  • the middle and bottom panels depict the results of MTT and colony formation assays of the tumor cells transfected with si-ADAMTS18s or control siRNAs.
  • Fig. 3 depicts the effects of exogenous ADAMTS18 on cell growth:
  • transient expression of exogenous ADAMTS18 in COS-7 cells detected by Western-blotting is depicted in the left panels.
  • the results of MTT assays of COS-7 cells transfected with ADAMTS18 cDNA or control siRNA are depicted in the right panel.
  • Fig. 3 depicts the effects of exogenous ADAMTS18 on cell growth:
  • Part B the effect of exogenous ADAMTS18 on cell growth is depicted.
  • Transient expression of exogenous ADAMTS18 in A549 cells detected by Western-blotting is depicted in the left panels and the results of MTT assays of A549 cells transfected with ADAMTS18 cDNA or control siRNA are depicted in the right panel.
  • Fig. 4 depicts the post-translational modification, subcellular localization and secretion into culture medium of exogenous ADAMTS18 protein:
  • Part A the results of western-blotting of exogenous ADAMTS18 protein after incubation with N-glycosidase are depicted.
  • Part B the subcellular localization of exogenous ADAMTS18 protein in COS-7 cells detected by ANTI-FLAG co-immunostained with DAPI is depicted.
  • Fig. 4 depicts the post-translational modification, subcellular localization and secretion into culture medium of exogenous ADAMTS18 protein:
  • Part C the expression of ADAMTS18 in COS-7 cells detected by semiquantitative RT-PCR analysis is depicted.
  • Part D the secretion of exogenous ADAMTS18 protein into culture medium, detected by Western-blot analysis is depicted.
  • Fig. 5 depicts the enhancement of cellular invasiveness induced by the introduction of ADAMTS18 into mammalian cells and MMP activity of exogenous or endogenous ADAMTS18:
  • Part A the result of assays demonstrating the invasive nature of COS-7 cells in Madrigal matrix after transfection of ADAMTS18-expressing plasmids are depicted. Giemsa staining (magnification, x100; lower panels) and the relative number of cells invading through the Matrigel-coated filters (upper panel) are shown. Assays were done twice and in duplicate wells.
  • Fig. 5 depicts the enhancement of cellular invasiveness induced by the introduction of ADAMTS18 into mammalian cells and MMP activity of exogenous or endogenous ADAMTS18:
  • Part B the results of cell growth assays, performed at the same time with Matrigel assays, are depicted.
  • Part D the suppression of the MMP activity of endogenous ADAMTS18 in DMS114 cells by si-ADAMTS18#1 is depicted.
  • an isolated or purified antibody refers to antibodies that are substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • polypeptide When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel.
  • SDS sodium dodecyl sulfate
  • antibodies and polypeptides of the present invention are isolated or purified.
  • nucleic acid molecule such as a cDNA molecule
  • a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding antibodies of the present invention are isolated or purified.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
  • modified R group or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids. Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • nucleic acid molecules are used interchangeably unless otherwise specifically indicated and are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.
  • the polynucleotide, oligonucleotide, nucleotides, nucleic acids, or nucleic acid molecules may be composed of DNA, RNA or a combination thereof.
  • cancer refers to cancer over-expressing the ADAMTS18 gene, in particular, lung cancer, including adenocarcinoma (ADC), squamous-cell carcinoma (SCC), large-cell carcinoma (LCC), and small-cell lung cancer (SCLC) and, esophageal cancer.
  • ADC adenocarcinoma
  • SCC squamous-cell carcinoma
  • LCC large-cell carcinoma
  • SCLC small-cell lung cancer
  • double-stranded molecule refers to a nucleic acid molecule that inhibits expression of a target gene, including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • double-stranded molecule is also referred to as “double-stranded nucleic acid ", “ double-stranded nucleic acid molecule”, “double-stranded polynucleotide”, “double-stranded polynucleotide molecule”, “double-stranded oligonucleotide” and “double-stranded oligonucleotide molecule”.
  • target sequence refers to a nucleotide sequence within mRNA or cDNA sequence of a target gene, which will result in suppression of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene.
  • a nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded molecule including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene.
  • a sense strand sequence of a double-stranded cDNA i.e., a sequence that mRNA sequence is converted into DNA sequence
  • a double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
  • the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule.
  • RNA region when a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base “t”s within the RNA region is replaced with base “u”s.
  • base "u"s within the DNA region is replaced with "t”s.
  • a sequence corresponding to a target sequence is "5'-GCCAGUAUCUCAAGAAATT-3'" (for SEQ ID NO: 11) or "5'-GGGCACAACUTTGGUATGA-3'” (for SEQ ID NO: 12).
  • a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
  • a complementary sequence to a target sequence is 3'- CGGUCAUAGAGTTCTTTAA -5'" (for SEQ ID NO: 11) or "3'- CCCGUGUUGAAACCATACT -5'" (for SEQ ID NO: 12).
  • the sequence corresponding to a target sequence shown in SEQ ID NO: 11 or 12 is the RNA sequence of SEQ ID NO: 11 or 12
  • the complementary sequence corresponding to a target sequence shown in SEQ ID NO: 11 or 12 is the RNA sequence of "3'- CGGUCAUAGAGUUCUUUAA-5'" (for SEQ ID NO:11) or "3'- CCCGUGUUGAAACCAUACU-5'" (for SEQ ID NO:12).
  • a double-stranded molecule may have one or two 3' overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
  • siRNA refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA includes a sense nucleic acid sequence (also referred to as “sense strand”), an antisense nucleic acid sequence (also referred to as “antisense strand”) or both.
  • the siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin.
  • the siRNA may either be a dsRNA or shRNA.
  • dsRNA refers to a construct of two RNA molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or “antisense” RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
  • shRNA refers to an siRNA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • siD/R-NA refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA.
  • a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used.
  • the siD/R-NA includes a sense nucleic acid sequence (also referred to as "sense strand”), an antisense nucleic acid sequence (also referred to as “antisense strand”) or both.
  • the siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin.
  • the siD/R-NA may either be a dsD/R-NA or shD/R-NA.
  • the term "dsD/R-NA” refers to a construct of two molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or "antisense” polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene.
  • One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
  • shD/R-NA refers to an siD/R-NA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • an "isolated nucleic acid” is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state.
  • examples of isolated nucleic acid include DNA, RNA, and derivatives thereof.
  • ADAMTS18 polynucleotide and ADAMTS18 polypeptide The present invention is based in part on the discovery of elevated expression of the ADAMTS18 gene in lung and esophageal cancer cells obtained from diseased patients.
  • the nucleotide sequence of the human ADAMTS18 gene is shown in SEQ ID NO: 1 and is also available as GenBank Accession No. NM_199355.
  • the ADAMTS18 gene encompasses the human ADAMTS18 gene as well as those of other animals including, but not limited to, non-human primate, mouse, rat, dog, cat, horse, and cow, and further includes allelic mutants and genes found in other animals as corresponding to the ADAMTS18 gene.
  • ADAMTS18 The amino acid sequence encoded the human ADAMTS18 gene is shown in SEQ ID NO: 2 and is also available as GenBank Accession No. NP_955387.1.
  • the polypeptide encoded by the ADAMTS18 gene is referred to as "ADAMTS18", and sometimes as “ADAMTS18 polypeptide” or "ADAMTS18 protein”.
  • a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptides that retain the biological ability of the ADAMTS18 protein may be used as such functional equivalents of each protein in the present invention.
  • the biological activities of the ADAMTS18 protein include, for example, regulating activity for cell differentiation and cancer cell proliferation activity (cancer cell proliferation enhancing activity). Further, the biological activity of the ADAMTS18 protein may include N-glycosylation activity, invasive activity and MMP (Matrix metalloproteinase) activity.
  • polypeptide may be one that includes an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the ADAMTA18 protein (e.g., SEQ ID NO: 2),, more preferably at least about 90% to 95% homology, even more preferably 96%, 97%, 98% or 99% homology.
  • the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the naturally occurring nucleotide sequence of the ADAMTS18 gene.
  • stringent (hybridization) conditions refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times of background, preferably 10 times of background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.
  • a condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the human ADAMTS18 protein can be routinely selected by a person skilled in the art.
  • hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer.
  • the following washing step can be conducted, for example, in a low stringent condition.
  • An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, preferably 50 degrees C, 2x SSC, 0.1% SDS.
  • High stringency conditions are often preferably used.
  • An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min.
  • factors such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
  • modifications of one, two, or more amino acids in a protein will not influence the function of the protein.
  • mutated or modified proteins i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition
  • mutated or modified proteins have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)).
  • the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in the human ADAMTS18 sequence.
  • the number of amino acid mutations is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 5 or 6 amino acids or less, and even more preferably 3 or 4 amino acids or less.
  • An amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution).
  • properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W).
  • A, I, L, M, F, P, W, Y, V hydrophilic
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspargine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
  • Such conservatively modified polypeptides are included in the present ADAMTS18 protein.
  • the present invention is not restricted thereto and the ADAMTS18 protein includes non-conservative modifications so long as they retain at least one biological activity of the ADAMTS18 protein.
  • the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
  • the ADAMTS18 gene of the present invention encompasses polynucleotides that encode such functional equivalents of the ADAMTS18 protein.
  • Diagnosing cancer II-1 Method for diagnosing cancer or a predisposition for developing cancer
  • the expression of the ADAMTS18 gene was found to be specifically elevated in patients with cancer, more particularly, in lung and esophageal cancer cells. Accordingly, the ADAMTS18 gene, as well as its transcription and translation products, find diagnostic utility as a marker for a cancer overexpressing ADAMTS18 gene, such as lung and esophageal cancer.
  • a cancer overexpressing ADAMTS18 gene such as lung and esophageal cancer
  • by measuring the expression of the ADAMTS18 gene in as subject-derived biological sample, such as a cell sample lung cancer or esophageal cancer can be diagnosed or detected. Such diagnosis or detection may be performed by comparing the expression level of ADAMTS18 gene between a subject-derived sample and a normal sample.
  • the present invention provides a method for detecting or diagnosing cancer and/or a predisposition for developing cancer in a subject by determining the expression level of the ADAMTS18 gene in the subject-derived biological sample.
  • Preferred cancers to be diagnosed or detected by the present method include lung and esophageal cancer.
  • lung cancer includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).
  • NSCLC includes adenocarcinoma, squamous cell carcinoma (SCC) and large-cell carcinoma.
  • the present invention provides a method for detecting or identifying cancer cells in a subject-derived lung or esophageal tissue sample, the method including the step of determining the expression level of the ADAMTS18 gene in a subject-derived tissue sample, wherein an increase in said expression level as compared to a normal control level indicates the presence or suspicion of cancer cells in the tissue.
  • the present invention may provide a doctor with useful information to diagnose that the subject suffers from the disease. For example, according to the present invention, when the suspicion or doubt of the presence of cancer cells in the tissue obtained from a subject is indicated, clinical decisions would be made by a doctor with consideration of this observation and another aspect including the pathological finding of the tissue, levels of known tumor marker(s) in blood, or clinical course of the subject, etc.
  • diagnostic lung cancer markers in blood include ACT, BFP, CA19-9, CA50, CA72-4, CA130, CA602, CEA, IAP, KMO-1, SCC, SLX, SP1, Span-1, STN, TPA, and cytokeratin 19 fragment.
  • diagnostic esophageal tumor markers in blood such as CEA, DUPAN-2, IAP, NSE, SCC, SLX and Span-1 are also well known. Namely, in a particular embodiment, according to the present invention, an intermediate result for examining the condition of a subject may also be provided.
  • the present invention provides a method for detecting a diagnostic marker of cancer, the method including the step of detecting the expression of the ADAMTS18 gene in a subject-derived biological sample as a diagnostic marker of cancer.
  • a diagnostic marker of cancer Preferable cancers to be diagnosed by the present method include lung cancer and esophageal cancer.
  • diagnosis is intended to encompass predictions and likelihood analysis.
  • the present method is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance for cancer. According to the present invention, an intermediate result for examining the condition of a subject may also be provided.
  • Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to determine that a subject suffers from the disease.
  • the present invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
  • a subject to be diagnosed by the present method is preferably a mammal.
  • exemplary mammals include, but are not limited to, human, non-human primate, mouse, rat, dog, cat, horse, and cow. It is preferred to collect a biological sample from the subject to be diagnosed to perform the diagnosis. Any biological material can be used as the biological sample for the determination so long as it can include the objective transcription or translation product of the ADAMTS18 gene due to cancer.
  • the biological samples include, but are not limited to, bodily tissues and fluids, such as blood, sputum, and urine.
  • the biological sample contains a cell population including an epithelial cell, more preferably a lung or esophageal epithelial cell derived from tissue suspected to be cancerous.
  • the cells may be purified from the obtained bodily tissues and fluids, and then used as the biological sample.
  • biological sample may be a tissue sample collected from an area suspected to be cancerous.
  • the tissue sample may be a lung tissue sample or esophageal tissue sample.
  • the expression level of the ADAMTS18 gene is determined in a subject-derived biological sample.
  • the expression level can be determined at the transcription product (i.e., mRNA) level, using methods known in the art.
  • the mRNA of the ADAMTS18 gene may be quantified using probes by hybridization methods (e.g., Northern hybridization).
  • the detection may be carried out on a filter, a chip or an array.
  • the use of an array is preferable for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including the ADAMTS18 gene.
  • Those skilled in the art can prepare such probes utilizing the sequence information of the ADAMTS18 gene.
  • the cDNA of the ADAMTS18 gene may be used as the probes.
  • the probe may be labeled with a suitable label, such as dyes and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • the transcription product of the ADAMTS18 gene may be quantified using primers by amplification-based detection methods (e.g., RT-PCR).
  • primers can also be prepared based on the available sequence information of the gene.
  • the primers used in the Example (SEQ ID NOs: 3 and 4) may be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the ADAMTS18 gene.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees C lower than the thermal melting point (T m ) for a specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the probes or primers may be of specific sizes.
  • the sizes are selected from the group consisting of at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides and the probes and primers may range in size from 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides and 25-30 nucleotides.
  • the translation product (i.e., protein) of the ADAMTS18 gene may be detected for the diagnosis or detection of the present invention.
  • the quantity of the ADAMTS18 protein may be determined.
  • a method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody may be monoclonal or polyclonal.
  • any antibody fragments or modified antibodies e.g., chimeric antibody, scFv, Fab, F(ab') 2 , Fv, etc.
  • any antibody fragments or modified antibodies e.g., chimeric antibody, scFv, Fab, F(ab') 2 , Fv, etc.
  • any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be observed via immunohistochemical analysis using an antibody against the ADAMTS18 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of the ADAMTS18 gene.
  • the translation product may be detected based on its biological activity.
  • the ADAMTS18 protein was demonstrated herein to be involved in the growth of cancer cells.
  • the cancer cell growth promoting ability of the ADAMTS18 protein may be used as an index of the ADAMTS18 protein existing in the biological sample.
  • cell growth promoting ability is also referred to as "cell proliferative activity” or "cell proliferation enhancing activity”.
  • the expression level of other cancer-associated genes for example, genes known to be differentially expressed in lung or esophageal cancer, may also be determined to improve the accuracy of the diagnosis.
  • the methods of the present invention compare the expression level of the ADAMTS18 gene in a subject-derived biological sample with that of a "control level".
  • control level refers to the expression level of the ADAMTS18 gene detected in a control sample and encompasses both a normal control level and a cancer control level.
  • normal control level refers to a level of the ADAMTS18 gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer.
  • a normal individual is one with no clinical symptom of lung and esophageal cancer.
  • a normal control level can be determined using a normal cell obtained from a non-cancerous tissue.
  • a "normal control level” may also be the expression level of the ADAMTS18 gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from lung cancer or esophageal cancer.
  • cancer control level refers to an expression level of the ADAMTS18 gene detected in the cancerous tissue or cell of an individual or population suffering from lung or esophageal cancer.
  • an increase in the expression level of the ADAMTS18 gene detected in a subject-derived sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing lung cancer or esophageal cancer.
  • the subject-derived sample may be any tissues obtained from test subjects, e.g., patients known to have or suspected of having cancer.
  • tissues may include epithelial cells. More particularly, tissues may be cancerous epithelial cells.
  • the expression level of the ADAMTS18 gene in a sample can be compared to a cancer control level of the ADAMTS18 gene. A similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer.
  • gene expression levels are deemed to be "increased" when the gene expression increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level.
  • the expression level of cancer marker genes including the ADAMTS18 gene in a biological sample can be considered to be increased if it increases from the normal control level of the corresponding cancer marker gene by, for example, 10% or more, 25% or more, or 50% or more; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the expression level of the target gene can be determined by detecting, e.g., determined by the hybridization intensity of nucleic acid probes to gene transcripts in a sample.
  • the control level may be determined at the same time with a test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the ADAMTS18 gene in samples from subjects whose disease state are known.
  • the control level can be a database of expression patterns from previously tested cells.
  • the expression level of the ADAMTS18 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples.
  • control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample.
  • standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.
  • a control level determined from a biological sample that is known to be non-cancerous is called “normal control level”.
  • the control level is determined from a cancerous biological sample, it will be called “cancerous control level”.
  • the expression level of the ADAMTS18 gene when the expression level of the ADAMTS18 gene is increased as compared to a normal control level or is similar to a cancerous control level, the subject may be diagnosed to be suffering from or at a risk of developing cancer.
  • a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
  • Difference between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes. Genes whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1. Furthermore, the present invention provides the use of the ADAMTS18 gene as cancerous markers. The ADAMTS18 gene are particularly useful for lung and esophageal cancerous markers.
  • a biological sample contains cancerous cells, especially lung or esophageal cancerous cells, by detecting the expression level of the ADAMTS18 gene as cancerous markers.
  • increasing the expression level of the ADAMTS18 gene in a biological sample as compared to a normal control level indicates that the biological sample contains cancerous cells.
  • the expression level of the ADAMTS18 gene can be determined by detecting the transcription or translation products of the gene as described above.
  • the ADAMTS18 gene differentially expressed between normal and cancerous cells also allow for the course of cancer treatment to be monitored, and the above-described method for diagnosing cancer can be applied for assessing the efficacy of a treatment on cancer.
  • the efficacy of a treatment on cancer can be assessed by determining the expression level of the ADAMTS18 gene in a cell(s) derived from a subject undergoing the treatment. If desired, test cell populations are obtained from the subject at various time points, before, during, and/or after the treatment.
  • the expression level of the ADAMTS18 gene can be, for example, determined following the method described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer. In the context of the present invention, it is preferable that the control level to which the detected expression level is compared be obtained from the ADAMTS18 gene expression in a cell(s) not exposed to the treatment of interest.
  • the expression level of the ADAMTS18 gene is compared to a control level that is obtained from a normal cell or a cell population containing no cancer cell, a similarity in the expression level indicates that the treatment of interest is efficacious and a difference in the expression level indicates less favorable clinical outcome or prognosis of that treatment.
  • a similarity in the expression level indicates that the treatment of interest is efficacious and a difference in the expression level indicates less favorable clinical outcome or prognosis of that treatment.
  • a difference in the expression level indicates efficacious treatment, while a similarity in the expression level indicates less favorable clinical outcome or prognosis.
  • the expression levels of the ADAMTS18 gene before and after a treatment can be compared according to the present method to assess the efficacy of the treatment.
  • the expression level detected in a subject-derived biological sample after a treatment i.e., post-treatment level
  • the expression level detected in a biological sample obtained prior to treatment onset from the same subject i.e., pre-treatment level.
  • a decrease in the post-treatment level compared to the pre-treatment level indicates that the treatment of interest is efficacious while an increase in or similarity of the post-treatment level to the pre-treatment level indicates less favorable clinical outcome or prognosis.
  • the term "efficacious" indicates that the treatment leads to a reduction in the expression of a pathologically up-regulated gene, an increase in the expression of a pathologically down-regulated gene or a decrease in size, prevalence, or metastatic potential of carcinoma in a subject.
  • "efficacious” means that the treatment retards or prevents the forming of tumor or retards, prevents, or alleviates at least one clinical symptom of cancer.
  • Assessment of the state of tumor in a subject can be made using standard clinical protocols.
  • efficaciousness of a treatment can be determined in association with any known method for diagnosing cancer. Cancers can be diagnosed, for example, by identifying symptomatic anomalies, e.g., weight loss, abdominal pain, back pain, anorexia, nausea, vomiting and generalized malaise, weakness, and jaundice.
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g., reducing the proliferation and metastasis of tumors.
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis includes 10%, 20%, 30% or more reduction, or stable disease.
  • kits and reagents The present invention also provides reagents for detecting or diagnosing cancer, i.e., reagents that can detect the transcription or translation product of the ADAMTS18 gene.
  • reagents include those capable of: (a) detecting mRNA of the ADAMTS18 gene; (b) detecting the ADAMTS18 protein; and/or (c) detecting the biological activity of the ADAMTS18 protein, in a subject-derived biological sample.
  • Suitable reagents include nucleic acids that specifically bind to or identify a transcription product of the ADAMTS18 gene.
  • a nucleic acid that specifically binds to or identifies a transcription product of the ADAMTS18 gene includes, for example, oligonucleotides (e.g., probes and primers) having a sequence that is complementary to a portion of the ADAMTS18 gene transcription product.
  • oligonucleotides are exemplified by primers and probes that are specific to the mRNA of the gene of interest and may be prepared based on methods well known in the art.
  • antibodies can be exemplified as reagents for detecting the translation product of the gene.
  • the probes, primers, and antibodies described above under the item of 'II-1 can be mentioned as suitable examples of such reagents.
  • These reagents may be used for the above-described diagnosis or detection of cancer, particularly lung cancer and esophageal cancer.
  • the assay format for using the reagents may be Northern hybridization or sandwich ELISA, both of which are well-known in the art.
  • the detection reagents may be packaged together in the form of a kit.
  • the detection reagents may be packaged in separate containers.
  • the detection reagents may be packaged with other reagents necessary for the detection.
  • a kit may include a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix) as the detection reagent, a control reagent (positive and/or negative), and/or a detectable label.
  • a kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes. These reagents and such may be retained in a container with a label.
  • Suitable containers include bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials, such as glass or plastic.
  • Instructions e.g., written, tape, VCR, CD-ROM, etc.
  • the present kit is suited for the detection and diagnosis of lung and esophageal cancer, it may also be useful in assessing the prognosis of cancer and/or monitoring the efficacy of a cancer therapy.
  • the reagents for diagnosing or detecting cancer may be immobilized on a solid matrix, such as a porous strip, to form at least one site for detecting cancer.
  • the measurement or detection region of the porous strip may include a plurality of sites, each containing a detection reagent (e.g., nucleic acid).
  • a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a separate strip from the test strip.
  • the different detection sites may contain different amounts of immobilized detection reagents (e.g., nucleic acid), i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the expression level of the ADAMTS18 gene in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • ADAMTS18 a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene
  • Screening methods Using the ADAMTS18 gene, a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene, it is possible to screen substances that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene.
  • Such substances may be used as pharmaceuticals for treating or preventing cancer, in particular, lung and esophageal cancer.
  • the present invention provides methods of screening for candidate substances for treating or preventing cancer using the ADAMTS18 gene, a polypeptide encoded by the gene or fragments thereof, or a transcriptional regulatory region of the gene.
  • a substance isolated by the screening method of the present invention is a substance that is expected to inhibit the expression of the ADAMTS18 gene, or the activity of the translation product of the gene, and thus, is a candidate for treating or preventing diseases attributed to, for example, cell proliferative diseases, such as cancer (in particular, lung and esophageal cancer). Namely, the substances screened through the present methods are deemed to have a clinical benefit and can be further tested for its ability to prevent cancer cell growth in animal models or test subjects.
  • substances to be identified through the present screening methods may be any compound or composition including several compounds.
  • a test substance exposed to a cell or protein in the screening methods of the present invention may be a single compound or a combination of compounds.
  • the compounds may be contacted sequentially or simultaneously.
  • test substances for example, cell extracts, cell culture supernatants, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention.
  • Test substances useful in the screenings described herein can also be antibodies that specifically bind to a protein of interest or a partial peptide thereof that lacks the biological activity of the original proteins in vivo.
  • Test substances of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including: (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection.
  • test substance libraries are well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided. It is herein revealed that suppression of the expression level and/or biological activity of ADAMTS18 lead to suppression of the growth of cancer cells. Therefore, when a substance suppresses the expression and/or activity of ADAMTS18, such suppression is indicative of a potential therapeutic effect in a subject.
  • a potential therapeutic effect refers to a clinical benefit with a reasonable expectation.
  • Examples of such clinical benefits include but are not limited to: (a) reduction in expression of the ADAMTS18 gene, (b) a decrease in size, prevalence, or metastatic potential of the cancer in the subject, (c) preventing cancers from forming, or (d) preventing or alleviating a clinical symptom of cancer.
  • test substance libraries are facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of ADAMTS18 protein.
  • One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.
  • Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule.
  • the three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • putative inhibitor Once a putative inhibitor has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below.
  • the resulting library of putative inhibitors may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, particularly lung and esophageal cancer.
  • Combinatorial Chemical Synthesis Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening.
  • simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library.
  • An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
  • Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6).
  • peptide libraries see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No.
  • WO 91/19735 encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J.
  • Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 106 -108 chemical entities).
  • a second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al.
  • the screened test substance is a protein
  • for obtaining a DNA encoding the protein either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein.
  • the obtained DNA finds use in preparing the test substance which is a candidate for treating or preventing cancer.
  • the expression of the ADAMTS18 gene is crucial for the growth and/or survival of cancer cells, in particular lung and esophageal cancer cells.
  • the ADAMTS18 protein has been demonstrated to have N-glycosylation activity, invasive activity as well as MMP (Matrix metalloproteinase) activity in the present invention. Accordingly, substances that suppress the function of the ADAMTS18 polypeptide would be presumed to inhibit the growth and/or survival of cancer cells, and therefore find use in treating or preventing cancer.
  • the present invention provides methods of screening a candidate substance for treating or preventing cancer, using the ADAMTS18 polypeptide. Further, the present invention also provides methods of screening a candidate substance for inhibiting the growth and/or survival of cancer cells, using the ADAMTS18 polypeptide.
  • fragments of the polypeptides may be used for the present screening, so long as it retains at least one biological activity of the naturally occurring ADAMTS18 polypeptide.
  • a fragment of the ADAMTS18 polypeptide may include a metalloproteinase domain of the polypeptide (e.g., 293 to 494 of SEQ ID NO: 2).
  • mature polypeptide of the ADAMTS18 e.g., 285 to 1221 of SEQ ID NO: 2
  • the ADAMTS18 protein may be produced in vitro by means of an in vitro translation system.
  • the ADAMTS18 polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.
  • the polypeptides or fragments used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence.
  • conventional peptide synthesis methods that can be adopted for the synthesis include: 1) Peptide Synthesis, Interscience, New York, 1966; 2) The Proteins, Vol.
  • the proteins may be obtained through any known genetic engineering methods for producing polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
  • a suitable vector including a polynucleotide encoding the objective protein in an expressible form e.g., downstream of a regulatory sequence including a promoter
  • the host cell is cultured to produce the protein.
  • a gene encoding the ADAMTS18 polypeptide is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.
  • a promoter may be used for the expression. Any commonly used promoters may be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3.
  • the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SRalpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such.
  • the introduction of the vector into host cells to express the ADAMTS18 gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.
  • electroporation method Chou et al., Nucleic Acids Res 1987, 15:1311-26
  • the calcium phosphate method Choen et al., Mol Cell Biol 1987, 7:2745-52
  • polypeptides or fragments thereof may be further linked to other substances, so long as the polypeptides and fragments retain at least one biological activity.
  • Usable substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. Such modifications may be used to confer additional functions or to stabilize the polypeptide and fragments.
  • the polypeptides may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide.
  • epitope-antibody system a commercially available epitope-antibody system may be used (Experimental Medicine 13: 85-90 (1995)).
  • Vectors that are capable of expressing a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and so on, by the use of its multiple cloning sites are commercially available.
  • a fusion protein prepared by introducing only small epitopes composed of several to a dozen amino acids so as not to change the property of the original polypeptide by the fusion, is also provided herein.
  • Epitopes such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and antibodies recognizing them may be used as the epitope-antibody system for detecting the binding activity between the polypeptides (Experimental Medicine 13: 85-90 (1995)).
  • the present invention provides a method of screening a candidate substance for treating or preventing cancer, which includes steps of: a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof; b) detecting binding (or binding activity) between the polypeptide or fragment and the test substance; and c) selecting the test substance that binds to the polypeptide as a candidate substance for treating or preventing cancer.
  • the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing ADAMTS18 associated disease (e.g., cancer), using the ADAMTS18 polypeptide or fragments thereof including the steps as follows: a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof; b) detecting the binding (or binding activity) between the polypeptide or fragment and the test substance; and c) correlating the binding of b) with the therapeutic effect of the test substance or compound.
  • the potential therapeutic effect of a test substance or compound on treating or preventing cancer can also be evaluated or estimated.
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of: (a) contacting a test substance with a polypeptide encoded by a polynucleotide of ADAMTS18; (b) detecting the binding activity between the polypeptide and the test substance; and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance binds to the polypeptide.
  • the therapeutic effect may be correlated with the binding level to ADAMTS18 polypeptide or a functional fragment thereof.
  • the test substance when the test substance binds to ADAMTS18 polypeptide or a functional fragment thereof, the test substance may identified or selected as the candidate substance having the requisite therapeutic effect.
  • the test substance when the test substance does not bind to an ADAMTS18 polypeptide or a functional fragment thereof, the test substance may identified as the substance having no significant therapeutic effect.
  • ADAMTS18 reduces cancer cell growth.
  • candidate substances that binds to ADAMTS18 polypeptide candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
  • the binding of a test substance to the ADAMTS18 polypeptide may be, for example, detected by immunoprecipitation using an antibody against the polypeptide. Therefore, for the purpose for such detection, it is preferred that the ADAMTS18 polypeptide or fragments thereof used for the screening contains an antibody recognition site.
  • the antibody used for the screening may be one that recognizes an antigenic region (e.g., epitope) of the ADAMTS18 polypeptide. Preparation methods for such antibodies are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the ADAMTS18 polypeptide or a fragment thereof may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody at its N- or C-terminus. The specificity of the antibody has been revealed, to the N- or C- terminus of the polypeptide.
  • a commercially available epitope-antibody system can be used (Experimental Medicine 1995, 13:85-90).
  • Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and such by the use of its multiple cloning sites are commercially available and can be used for the present invention.
  • fusion proteins containing much smaller epitopes to be detected by immunoprecipitation with an antibody against the epitopes are also known in the art (Experimental Medicine 1995, 13:85-90).
  • epitopes composed of several to a dozen amino acids so as not to change the property of the ADAMTS18 polypeptide or fragments thereof, can also be used in the present invention.
  • Examples include polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), and such and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the ADAMTS18 polypeptide (Experimental Medicine 13: 85-90 (1995)).
  • His-tag polyhistidine
  • influenza aggregate HA human c-myc
  • FLAG Vesicular stomatitis virus glycoprotein
  • VSV-GP Vesicular stomatitis virus glycoprotein
  • T7-tag T7 gene 10 protein
  • HSV-tag human simple herpes virus glycoprotein
  • E-tag an epitope on monoclonal phage
  • Glutathione S-transferase is also well-known as the counterpart of the fusion protein to be detected by immunoprecipitation.
  • GST is used as the protein to be fused with the ADAMTS18 polypeptide or fragment thereof to form a fusion protein
  • the fusion protein can be detected either with an antibody against GST or a substance specifically binding to GST, i.e., such as glutathione (e.g., glutathione-Sepharose 4B).
  • an immune complex is formed by adding an antibody (recognizing the ADAMTS18 polypeptide or a fragment thereof itself, or an epitope tagged to the polypeptide or fragment) to the reaction mixture of the ADAMTS18 polypeptide and the test substance. If the test substance has the ability to bind the polypeptide, then the formed immune complex will consist of the ADAMTS18 polypeptide, the test substance, and the antibody. On the contrary, if the test substance is devoid of such ability, then the formed immune complex only consists of the ADAMTS18 polypeptide and the antibody. Therefore, the binding ability of a test substance to ADAMTS18 polypeptide can be examined by, for example, measuring the size of the formed immune complex.
  • an antibody recognizing the ADAMTS18 polypeptide or a fragment thereof itself, or an epitope tagged to the polypeptide or fragment
  • Any method for detecting the size of a compound can be used, including chromatography, electrophoresis, and such.
  • chromatography electrophoresis
  • Protein A or Protein G sepharose can be used for quantitating the formed immune complex.
  • SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Detection may be achieved using conventional staining method, such as Coomassie staining or silver staining, or, for proteins that is difficult to detect, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35 S-methionine or 35 S-cysteine, labeling proteins in the cells, and detecting the proteins.
  • the target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
  • the ADAMTS18 polypeptide or a fragment thereof used for the screening of substances that bind thereto may be bound to a carrier.
  • carriers that may be used for binding the polypeptides include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercially available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column. Alternatively, the use of magnetic beads is also known in the art, and enables to readily isolate polypeptides and substances bound on the beads via magnetism.
  • the binding of a polypeptide to a carrier may be conducted according to routine methods, such as chemical bonding and physical adsorption.
  • a polypeptide may be bound to a carrier via antibodies specifically recognizing the protein.
  • binding of a polypeptide to a carrier can also be conducted by means of interacting molecules, such as the combination of avidin and biotin. Screening using such carrier-bound ADAMTS18 polypeptide or fragments thereof include, for example, contacting a test substance to the carrier-bound polypeptide, incubating the mixture, washing the carrier, and detecting and/or measuring the substance bound to the carrier.
  • the binding may be carried out in buffer, for example, but are not limited to, phosphate buffer and Tris buffer, as long as the buffer does not inhibit the binding.
  • a carrier-bound ADAMTS18 polypeptide or fragments thereof, and a composition are used as the test substance in a screening method
  • a composition e.g., cell extracts, cell lysates, etc.
  • affinity chromatography e.g., affinity chromatography
  • the ADAMTS18 polypeptide may be immobilized on a carrier of an affinity column, and a test substance, containing a substance capable of binding to the polypeptides, is applied to the column. After loading the test substance, the column is washed, and then the substance bound to the polypeptide is eluted with an appropriate buffer.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention.
  • the interaction between the ADAMTS18 polypeptide and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of the polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide and test substance using a biosensor such as BIAcore.
  • a protein binding to the ADAMTS18 polypeptide can be obtained by preparing first a cDNA library from cells, tissues, organs, or cultured cells (e.g., PC cell lines) expected to express at least one protein binding to the ADAMTS18 polypeptide using a phage vector (e.g., ZAP), expressing the proteins encoded by the vectors of the cDNA library on LB-agarose, fixing the expressed proteins on a filter, reacting the purified and labeled ADAMTS18 polypeptide with the above filter, and detecting the plaques expressing proteins to which the ADAMTS18 polypeptide has bound according to the label of the ADAMTS18 polypeptide.
  • a cDNA library from cells, tissues, organs, or cultured cells (e.g., PC cell lines) expected to express at least one protein binding to the ADAMTS18 polypeptide using a phage vector (e.g., ZAP), expressing the proteins encoded by the vectors of the cDNA library
  • Labeling substances such as radioisotope (e.g., 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucosidase), fluorescent substances (e.g., fluorescein isothiocyanate (FITC), rhodamine) and biotin/avidin, may be used for the labeling of ADAMTS18 polypeptide in the present method.
  • radioisotope e.g., 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I
  • enzymes e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucosidase
  • fluorescent substances e.g., fluorescein isothiocyanate (FITC), rhodamine
  • the protein when it is labeled with an enzyme, it can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.
  • the ADAMTS18 polypeptide bound to the protein can be detected or measured by utilizing an antibody that specifically binds to the ADAMTS18 polypeptide or a peptide or polypeptide (for example, GST) that is fused to the ADAMTS18 polypeptide.
  • the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance.
  • the antibody against the ADAMTS18 polypeptide may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance.
  • the antibody bound to the ADAMTS18 polypeptide in the present screening may be detected or measured using protein G or protein A column.
  • Antibodies to be used in the present screening methods can be prepared using techniques well known in the art.
  • Antigens to prepared antibodies may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, rabbit, or rat, more preferably from a human.
  • the polypeptide used as the antigen can be recombinantly produced or isolated from natural sources.
  • the polypeptides to be used as an immunization antigen may be a complete protein or a partial peptide derived from the complete protein.
  • animals of the order Rodentia, Lagomorpha or Primate are used.
  • Animals of the Rodentia order include, for example, mice, rats and hamsters.
  • Animals of Lagomorpha order include, for example, hares, pikas, and rabbits.
  • Animals of Primate order include, for example, monkeys of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkeys, sacred baboons and chimpanzees.
  • antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc.
  • PBS phosphate buffered saline
  • the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals.
  • a standard adjuvant such as Freund's complete adjuvant
  • an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days.
  • An appropriate carrier may also be used for immunization.
  • the serum is examined by a standard method for an increase in the amount of desired antibodies.
  • Polyclonal antibodies may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method.
  • Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies isolated from the serum.
  • Immunoglobulin G or M can be prepared from a fraction which recognizes only the objective polypeptide using, for example, an affinity column coupled with the polypeptide, and further purifying this fraction using protein A or protein G column.
  • immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion.
  • the immune cells used for cell fusion are preferably obtained from spleen.
  • Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
  • the above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al., (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).
  • Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium).
  • HAT medium hyperxanthine, aminopterin, and thymidine containing medium.
  • the cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma, to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
  • human lymphocytes such as those infected by the EB virus, may be immunized with an antigen, cells expressing such antigen, or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the antigen (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).
  • JP-A Japanese Patent Application No.
  • the obtained hybridomas may be subsequently transplanted into the abdominal cavity of a mouse and the ascites may be extracted.
  • the obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column carrying an objective antigen.
  • Antibodies against the ADAMTS18 polypeptide can be used not only in the present screening method, but also for the detection of the polypeptides as cancer markers in biological samples as described in "II. Diagnosing cancer". They may further serve as candidates for agonists and antagonists of the polypeptides of interest. In addition, such antibodies, serving as candidates for antagonists, can be applied to the antibody treatment for diseases related to the ADAMTS18 polypeptide including lung and esophageal cancer as described infra.
  • Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)).
  • a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody.
  • an immune cell such as a hybridoma or an immunized lymphocyte producing the antibody
  • host cells such as a recombinant antibody.
  • Such recombinant antibody can also be used in the context of the present screening.
  • antibodies used in the screening and so on may be fragments of antibodies or modified antibodies, so long as they retain the original binding activity.
  • the antibody fragment may be an Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)).
  • an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin.
  • a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
  • An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). Modified antibodies can be obtained through chemically modification of an antibody. These modification methods are conventional in the field. Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by appropriately selected and combined column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)); however, the present invention is not limited thereto.
  • a protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
  • Exemplary chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)).
  • the chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
  • two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton et al., Cell 1992, 68:597-612” and “Fields et al., Trends Genet 1994, 10:286-92").
  • an ADAMTS18 polypeptide or a fragment thereof is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • a cDNA library is prepared from cells expected to express at least one protein binding to the ADAMTS18 polypeptide such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region.
  • the cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the ADAMTS18 polypeptide is expressed in the yeast cells, the binding of the two activates a reporter gene, making positive clones detectable).
  • a protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
  • reporter genes include, but are not limited, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
  • the substances isolated by this screening are considered to candidates for agonists or antagonists of the ADAMTS18 polypeptide.
  • the term "agonist” refers to molecules that activate the function of the polypeptide by binding thereto.
  • the term “antagonist” refers to molecules that inhibit the function of the polypeptide by binding thereto.
  • an substance isolated by this screening as an antagonist is a candidate that inhibits the in vivo interaction of the ADAMTS18 polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).
  • the present invention also provides a method for screening a candidate substance for treating or preventing cancer using the ADAMTS18 polypeptide or fragments thereof including the steps as follows: a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof; b) detecting the biological activity of the polypeptide or fragment of the step (a); and c) selecting the test substance that reduces the biological activity of the polypeptide as compared to the biological activity in the absence of the test substance.
  • the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing an ADAMTS18 associated disease may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing an ADAMTS18 associated disease, using the ADAMTS18 polypeptide or fragments thereof including the steps as follows: a) contacting a test substance with an ADAMTS18 polypeptide or a functional fragment thereof; b) detecting the biological activity of the polypeptide or fragment of step (a); and c) correlating the biological activity of b) with the therapeutic effect of the test substance.
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of: (a) contacting a test substance with a polypeptide encoded by a polynucleotide of ADAMTS18 gene; (b) detecting the biological activity of the polypeptide of step (a); and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the polypeptide encoded by the polynucleotide of ADAMTS18 gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.
  • the therapeutic effect may be correlated with the biological activity of an ADAMTS18 polypeptide or a functional fragment thereof.
  • the test substance when the test substance suppresses or inhibits the biological activity of an ADAMTS18 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not suppress or inhibit the biological activity of ADAMTS18 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • ADAMTS18 reduces cancer cell growth.
  • candidate substances that suppresses the biological activity of the polypeptide candidate substances that have the potential to treat or prevent cancers can be identified.
  • Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance binding to ADAMTS18 protein inhibits described above activities of the cancer, it may be concluded that such substance has the ADAMTS18 specific therapeutic effect.
  • any substance can be used for the screening so long it suppresses or reduces a biological activity of the ADAMTS18 polypeptide.
  • the phrase "suppress or reduce a biological activity” encompasses at least 10% suppression of the biological activity of ADAMTS18 in comparison with in the absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at least 90% suppression. Such suppression can serve an index in the present screening method.
  • control cells which do not express ADAMTS18 polypeptide are used.
  • the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing an ADAMTS18 associated disease, using the ADAMTS18 polypeptide or fragments thereof including the steps as follows: a) culturing cells which express a ADAMTS18 polypeptide or a functional fragment thereof, and control cells that do not express a ADAMTS18 polypeptide or a functional fragment thereof in the presence of the test substance; b) detecting the biological activity of the cells which express the protein and control cells; and c) selecting the test compound that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance.
  • the ADAMTS18 polypeptide has been demonstrated to be required for the growth or viability of lung and esophageal cancer cells.
  • the biological activities of the ADAMTS18 polypeptide that can be used as an index for the screening include such cell growth promoting activity of the human ADAMTS18 polypeptide.
  • cell growth promoting ability is also referred to as "cell proliferative activity” or "cell proliferation enhancing activity”.
  • the ADAMTS18 polypeptide has N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity. Thus, these activity may also be used as indexes for the screening.
  • MMP Matrix Metalloproteinase
  • the biological activity to be detected in the present method is cell proliferation enhancing activity
  • it can be detected, for example, by preparing cells which express the ADAMTS18 polypeptide or a fragment thereof, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by detecting wound-healing activity, conducting Matrigel invasion assay and measuring the colony forming activity.
  • control cells that do not express the ADAMTS18 polypeptide are preferably used.
  • the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer, using the ADAMTS18 polypeptide or fragments thereof including the steps as follows: a) culturing cells which express an ADAMTS18 polypeptide or a functional fragment thereof in the presence of the test substance; b) detecting the biological activity of the cells which express the polypeptide; and c) selecting the test substance that inhibits the biological activity in the cells which express the polypeptide as compared to the proliferation detected in the absence of the test substance.
  • the biological activity to be detected in the present method is N-glycosylation activity
  • it can be detected, for example, by contacting a cell expressing ADAMTS18 polypeptide or functional equivalent thereof having N-glycosylation activity, or a purified ADAMTS18 polypeptide with a test substance, and detecting N-glycosylation level of the ADAMTS18 polypeptide or the functional equivalent under a suitable condition for N-glycosylation of the ADAMTS18 polypeptide.
  • a test substance that modulates glycosylation level of the ADAMTS18 polypeptide or functional equivalent is thereby identified.
  • N-glycosylation level of an ADAMTS18 polypeptide can be determined by methods known in the art. For example, N-glycosylation of the polypeptide may be detected by comparing the molecular weight. Molecular weight of an N-glycosylated protein is larger than that of predicted size calculated from the amino acid sequence of the polypeptide by addition of glycoside chain. Methods for estimating a molecular weight of a protein are well known.
  • radiolabeled donor for N-glycosylation may be used for detection the addition of glycoside chain to the polypeptide.
  • Transfer of the radiolabel to the ADAMTS18 protein can be detected, for example, by SDS-PAGE electrophoresis and fluorography.
  • the ADAMTS18 polypeptides can be separated from the glycosyl donor by filtration, and the amount of radiolabel retained on the filter quantitated by scintillation counting.
  • suitable labels that can be attached to glycosyl donor such as chromogenic and fluorescent labels, and methods of detecting transfer of these labels to the ADAMTS18 polypeptide, are known in the art.
  • N-glycosylation level of ADAMTS18 polypeptide can be determined by reagents that selectively recognize glycosylated site of the polypeptide. For example, after incubation of the ADAMTS18 polypeptide and a candidate substance, under the condition capable of glycosylation of the polypeptide, the N-glycosylation level of the polypeptide can be detected by immunological method. Any immunological techniques using an antibody that recognizes a glycosylated polypeptide can be used for the detection. For example, ELISA or Immunoblotting with antibodies recognizing glycosylated polypeptide can be used for the present invention.
  • glycosylated protein can be detected using reagents that selectively bind glycoside chain with high affinity.
  • reagents are known in the art.
  • lectins are well known as glycoside chain specific probe.
  • Lectin reagent conjugated with detectable label such as alkaline-phosphatase is commercially available.
  • the N-glycosylation level of a polypeptide in a cell may be estimated by separation of cell lysate. For example, SDS-polyacrylamide gel can be used as the separation of the polypeptide. The polypeptide separated in the gels is transferred to nitrocellulose membranes for immunoblotting analysis.
  • the biological activity to be detected in connection with the present method is invasive activity
  • it can be detected, for example, by preparing cells that express the ADAMTS18 polypeptide and counting invasive cells number using Matrigel invasion assay, for example, as shown in Fig. 5A.
  • the substances that reduce the invasive cell number are selected as candidate substance for treating or preventing lung or esophageal cancer.
  • the biological activity to be detected in connection with the present method is MMP activity
  • it can be detected, for example, by contacting a cell expressing ADAMTS18 polypeptide or functional equivalent thereof, or a purified ADAMTS18 polypeptide or functional equivalent thereof with a test substances, followed by applying MMP assay to evaluate the MMP activity.
  • the MMP assay can be conducted by methods well-known in the art.
  • the MMP assay may be conducted by using, a commercially available assay kit that includes a substrate for MMP.
  • the substance isolated by the present screening method is a candidate for an antagonist of the ADAMTS18 polypeptide, and thus, is a candidate that inhibits the in vivo interaction of the polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).
  • such screening may include, for example, the following steps: a) contacting a test substance with a cell expressing an ADAMTS18 gene; b) detecting the expression level of the ADAMTS18 gene; c) comparing the expression level with the expression level detected in the absence of the test substance; and d) selecting the test substance that reduces the expression level as a candidate substance for treating or preventing cancer.
  • the therapeutic effect of the test substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer.
  • such screening may include, for example, the following steps: a) contacting a test substance with a cell expressing an ADAMTS18 gene; b) detecting the expression level of the ADAMTS18 gene; and c) correlating the expression level of b) with the therapeutic effect of the test substance.
  • the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance in treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of: (a) contacting a candidate substance with a cell expressing ADAMTS18; and; (b) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance reduces the expression level of ADAMTS18 as compared to a control.
  • the therapeutic effect may be correlated with the expression level of the ADAMTS18 gene.
  • the test substance when the test substance reduces the expression level of the ADAMTS18 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression level of the ADAMTS18 gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • ADAMTS18 gene reduces cancer cell growth.
  • candidate substance that reduces the expression level of ADAMTS18 candidate substance that have the potential to treat or prevent cancers can be identified. Potential of these candidate substance to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
  • a substance that inhibits the expression of the ADAMTS18 gene can be identified by contacting a cell expressing the ADAMTS18 gene with a test substance and then determining the expression level of the ADAMTS18 gene. Naturally, the identification may also be performed using a population of cells that express the gene in place of a single cell. A decreased expression level detected in the presence of a test substance as compared to the expression level in the absence of the test substance indicates the test substance as being an inhibitor of the ADAMTS18 gene, suggesting the possibility that the test substance is useful for inhibiting cancer, thus the test substance to be used for the treatment or prevention of cancer.
  • the expression level of a gene can be estimated by methods well known to one skilled in the art.
  • the expression level of the ADAMTS18 gene can be, for example, determined following the method described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer'.
  • the cell or the cell population used for such identification may be any cell or any population of cells so long as it expresses the ADAMTS18 gene.
  • the cell or population may be or contain a lung and esophageal epithelial cell derived from a tissue.
  • the cell or population may be or contain an immortalized cell derived from a carcinoma cell, including lung and esophageal cancer cell.
  • Cells expressing the ADAMTS18 gene include, for example, cell lines established from cancers (e.g., lung and esophageal cancer cell lines such as NCI-H1781, NCI-H1373, LC319, A549, PC-14, SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170, LU61, TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, TE10, SBC-3, SBC-5, DMS114, DMS273 etc.).
  • the cell or population may be or contain a cell which has been transfected with the ADAMTS18 gene.
  • the present method allows screening of various test substances mentioned above and is particularly suited for screening functional nucleic acid molecules including antisense RNA, siRNA, and such.
  • the present invention provides a method that includes the following steps of: a) contacting a test substance with a cell into which a vector, including a transcriptional regulatory region of an ADAMTS18 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; b) detecting the expression or activity of said reporter gene; c) comparing the expression level or activity with the expression level or activity detected in the absence of the substance ; and d) selecting the substance that reduces the expression or activity of said reporter gene as a candidate substance for treating or preventing cancer.
  • the therapeutic effect of the test substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer.
  • the present invention provides a method which includes the following steps of: a) contacting a test substance with a cell into which a vector, composed of a transcriptional regulatory region of an ADAMTS18 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; b) detecting the expression or activity of said reporter gene; and c) correlating the expression level of b) with the therapeutic effect of the test substance.
  • the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of: (a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of ADAMTS18 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) measuring the expression or activity of said reporter gene; and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression or activity of said reporter gene.
  • the therapeutic effect may be correlated with the expression or activity of said reporter gene.
  • the test substance when the test substance reduces the expression or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • ADAMTS18 gene reduces cell growth.
  • candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
  • the reporter construct required for the screening can be prepared using the transcriptional regulatory region of the ADAMTS18 gene, which can be obtained as a nucleotide segment containing the transcriptional regulatory region from a genome library based on the nucleotide sequence information of the gene.
  • the transcriptional regulatory region may be, for example, the promoter sequence of the ADAMTS18 gene.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of ADAMTS18 gene.
  • the transcriptional regulatory region of ADAMTS18 gene herein is the region from start codon to at least 500 bp upstream, preferably 1,000 bp, more preferably 5,000 or 10,000 bp upstream.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
  • an substance can be identified as inhibiting or enhancing the expression of the ADAMTS18 gene through detecting the expression level of the reporter gene product.
  • Illustrative reporter genes include, but are not limited to, luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa, Ade2 gene, HIS3 gene, and others well-known in the art. Methods for detection of the expression of these genes are well known in the art.
  • a vector containing a reporter construct may be infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on).
  • the phrase "reduces the expression or activity” encompasses at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the compound, more preferably at least 25%, 50% or 75% reduction and most preferably at 95% reduction.
  • ADAMTS18 gene differentially expressed between cancerous and non-cancerous cells disclosed herein allow for a putative therapeutic or prophylactic inhibitor of cancer to be tested in a test cell population from a selected subject in order to determine if the substance is a suitable inhibitor of cancer in the subject.
  • test cell populations contain cancer cells expressing the ADAMTS18 gene.
  • the test cell is a lung and esophageal epithelial cell.
  • a test cell population may be incubated in the presence of a candidate therapeutic substance or agent and the expression of the ADAMTS18 gene in the test cell population may be measured and compared to one or more reference profiles, e.g., a cancerous reference expression profile or a non-cancerous reference expression profile.
  • a decrease in the expression of the ADAMTS18 gene in a test cell population relative to a reference cell population containing cancer indicates that the substance has therapeutic potential.
  • a similarity in the expression of the ADAMTS18 gene in a test cell population relative to a reference cell population not containing cancer indicates that the substance has therapeutic potential.
  • compositions for treating or preventing cancer The substances screened by any of the screening methods of the present invention, antisense nucleic acids and double-stranded molecules (e.g., siRNA) against the ADAMTS18 gene, and antibodies against the ADAMTS18 polypeptide inhibit or suppress the expression of the ADAMTS18 gene, or the biological activity of the ADAMTS18 polypeptide and inhibit or disrupt cancer cell cycle regulation, cancer cell proliferation and cell invasion.
  • the present invention provides compositions for treating or preventing cancer, wherein the compositions include substances identified by any of the screening methods of the present invention, antisense nucleic acids or double-stranded molecules against the ADAMTS18 gene, or antibodies against the ADAMTS18 polypeptide.
  • the present compositions can be used for treating or preventing cancer, in particular, cancer such as lung and esophageal cancer.
  • compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.
  • suitable pharmaceutical formulations for the active ingredients of the present invention detailed below include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation.
  • administration is intravenous.
  • the formulations are optionally packaged in discrete dosage units.
  • compositions suitable for oral administration include capsules, microcapsules, cachets and tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, elixirs, granules, solutions, suspensions and emulsions.
  • the active ingredient is optionally administered as a bolus electuary or paste.
  • the pharmaceutical composition may be administered non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid.
  • the active ingredients of the present invention can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending substances, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation.
  • pharmaceutically acceptable carriers or media specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending substances, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation.
  • the amount of active ingredient contained in such a preparation makes a suitable dosage within the indicated range acquirable.
  • additives that can be admixed into tablets and capsules include, but are not limited to, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry.
  • a tablet may be made by compression or molding, optionally with one or more formulational ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine in which the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made via molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient in vivo. A package of tablets may contain one tablet to be taken on each of the month.
  • a liquid carrier such as oil
  • Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle prior to use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • sterile composites for injection can be formulated following normal drug implementations using vehicles, such as distilled water, suitable for injection.
  • vehicles such as distilled water, suitable for injection.
  • Physiological saline, glucose, and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injection.
  • adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride
  • solubilizers such as alcohol, for example, ethanol
  • polyalcohols such as propylene glycol and polyethylene glycol
  • non-ionic surfactants such as Polysorbate 80 (TM) and HCO-50.
  • Sesame oil or soybean oil can be used as an oleaginous liquid, which may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer, and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and/or an anti-oxidant.
  • a prepared injection may be filled into a suitable ampoule.
  • Formulations for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol.
  • Formulations for topical administration in the mouth include lozenges, which contain the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles including the active ingredient in a base such as gelatin, glycerin, sucrose or acacia.
  • a liquid spray or dispersible powder or in the form of drops may be used. Drops may be formulated with an aqueous or non-aqueous base also including one or more dispersing agents, solubilizing agents or suspending agents.
  • compositions are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray.
  • Pressurized packs may include a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compositions may take the form of a dry powder composition, for example, a powder mix of an active ingredient and a suitable powder base such as lactose or starch.
  • a powder mix of an active ingredient and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.
  • compositions include implantable devices and adhesive patches that release a therapeutic agent.
  • the above-described formulations adapted to give sustained release of the active ingredient, may be employed.
  • the pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
  • Preferred unit dosage formulations are those containing an effective dose, as recited under the item of 'VI. Method for treating or preventing cancer' (infra), of each active ingredients of the present invention or an appropriate fraction thereof.
  • compositions containing screened substances The present invention provides compositions for treating or preventing cancers including any of the substances identified by the above-described screening methods of the present invention.
  • a substances identified by the method of the present invention can be directly administered or can be formulated into a dosage form according to any conventional pharmaceutical preparation method detailed above.
  • Double-stranded molecules e.g., siRNA
  • double-stranded molecules against the ADAMTS18 gene can be used to reduce the expression level of the genes.
  • double-stranded molecule refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)) as described in "Definitions”.
  • double-stranded molecules include a sense nucleic acid sequence and an anti-sense nucleic acid sequence against the ADAMTS18 gene.
  • the double-stranded molecule is constructed so that it includes both a portion of the sense and complementary antisense sequences of the target gene (i.e., the ADAMTS18 gene), and may also be a single construct taking a hairpin structure, wherein the sense and antisense strands are linked via a single-strand.
  • the double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells.
  • the identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA.
  • a double-stranded molecule of the present invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the ADAMTS18 gene under stringent conditions.
  • target sequence or “target nucleic acid” or “target nucleotide”.
  • nucleotide sequence of the “target sequence” can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
  • a double-stranded molecule is preferably less than 500, 200, 100, 50, or 25 base pairs in length. More preferably, a double stranded molecule is 19-25 base pairs in length.
  • Exemplary target sequences of double-stranded molecules against the ADAMTS18 gene include the nucleotide sequences of SEQ ID NO: 11 and 12.
  • the present pharmaceutical composition may include a double-stranded RNA molecule (i.e., siRNA) including the nucleotide sequence 5'- GCCAGUAUCUCAAGAAAUU -3' (for SEQ ID NO: 11), and 5'- GGGCACAACUUUGGUAUGA -3' (for SEQ ID NO: 12) as the sense strand.
  • siRNA double-stranded RNA molecule
  • 3' overhangs can be added to the 3'end of the target sequence in the sense and/or antisense strand.
  • the number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5.
  • the added nucleotides form a single strand at the 3'end of the sense and/or antisense strand of the double-stranded molecule.
  • the nucleotides to be added is preferably "u" or "t", but are not limited to.
  • a loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense strands in order to form a hairpin loop structure.
  • the double-stranded molecule contained in the pharmaceutical composition of the present invention may take the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to [A]. .
  • the polynucleotide strand which includes a sequence corresponding to a target sequence may be referred to as "sense strand".
  • [A] is the sense strand
  • [B] is a single stranded polynucleotide consisting of 3 to 23 nucleotides
  • [A'] is a polynucleotide strand which includes the antisense strand containing a complementary sequence of a target sequence, specifically hybridizing to an mRNA or a cDNA of the ADAMTS18 gene (i.e., a sequence hybridizing to the target sequence of the sense strand [A]).
  • the polynucleotide strand which includes a complementary sequence to a target sequence, specifically hybridizing to an mRNA or a cDNA of the ADAMTS18 gene may be referred to as "antisense strand".
  • the region [A] hybridizes to [A'], and then a loop consisting of the region [B] is formed.
  • the loop sequence may be preferably 3 to 23 nucleotides in length.
  • the loop sequence for example, can be selected from a group consisting of following sequences (www.ambion.com/techlib/tb/tb_506.html): CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002, 418: 435-8.
  • UUCG Lee NS et al., Nature Biotechnology 2002, 20:500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003, 100(4):1639-44.
  • UUCAAGAGA Dykxhoorn DM et al., Nature Reviews Molecular Cell Biology 2003, 4:457-67.
  • 'UUCAAGAGA ttcaagaga" in DNA
  • loop sequence consisting of 23 nucleotides also provides an active siRNA (Jacque JM et al., Nature 2002, 418:435-8).
  • Exemplary hairpin siRNA suitable for the ADAMTS18 gene include: 5'-GCCAGUAUCUCAAGAAAUU -[b]-AAUUUCUUGAGAUACUGGC -3' (target sequence of SEQ ID NO: 11); 5'-AAUUUCUUGAGAUACUGGC -[b]-GCCAGUAUCUCAAGAAAUU -3' (target sequence of SEQ ID NO: 11) and; 5'-GGGCACAACUUUGGUAUGA-[b]- UCAUACCAAAGUUGUGCCC-3' (target sequence of SEQ ID NO: 12); 5'-UCAUACCAAAGUUGUGCCC-[b]- GGGCACAACUUUGGUAUGA-3' (target sequence of SEQ ID NO: 12).
  • nucleotide sequences of suitable double-stranded molecules for the present invention can be designed using an siRNA design computer program available from the Ambion website (www.ambion.com/techlib/ misc/siRNA_finder.html).
  • the computer program selects nucleotide sequences for double-stranded molecule synthesis based on the following protocol.
  • Target Sites for double-stranded molecules 1. Beginning with the AUG start codon of the object transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential target sites. Tuschl et al. Genes Cev 1999, 13(24):3191-7 don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 nucleotides) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex. 2.
  • the homology search can be performed using BLAST (Altschul SF et al., Nucleic Acids Res 1997, 25:3389-402; J Mol Biol 1990, 215:403-10.), which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/. 3. Select qualifying target sequences for synthesis. At Ambion, preferably several target sequences can be selected along the length of the gene to evaluate.
  • the double-stranded molecules of the present invention can be prepared using any chemical synthetic method known in the art. For example, according to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double-stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that includes the target sequence and antisense thereof is transcribed into the double-stranded molecule in vitro.
  • the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5).
  • the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down.
  • the annealed double-stranded polynucleotide can be purified by usually employed methods known in the art. Examples of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
  • the regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner.
  • the double-stranded molecules can be transcribed intracellularly by cloning ADAMTS18 gene template into a vector containing, e.g., an RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
  • snRNA small nuclear RNA
  • Standard techniques are known in the art for introducing a double-stranded molecule into cells.
  • a double-stranded molecule can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts.
  • the double-stranded molecules are typically modified as described below for antisense molecules.
  • Other modifications are also available, for example, cholesterol-conjugated double-stranded molecule has shown improved pharmacological properties (Song et al., Nature Med 2003, 9:347-51). These conventionally used techniques may also be applied for the double-stranded molecules contained in the present compositions.
  • a DNA encoding the double-stranded molecule may be carried in a vector (hereinafter, also referred to as 'siRNA vector') and the double-stranded molecule may be contained in the present composition in the form of vector which enables expression of the double-stranded molecule in vivo.
  • a vector hereinafter, also referred to as 'siRNA vector'
  • Such vectors may be produced, for example, by cloning a portion of the target sequence sufficient to inhibit the in vivo expression of the target gene into an expression vector having operatively-linked regulatory sequences (e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nature Biotechnology 2002, 20: 500-5).
  • operatively-linked regulatory sequences e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter
  • an RNA molecule that is antisense to mRNA of the target gene is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of the target gene is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate the double-stranded molecule construct for silencing the expression of the target gene.
  • the sense and antisense strands may be transcribed together with the help of one promoter.
  • the sense and antisense strands may be linked via a polynucleotide sequence to form a single-stranded construct having secondary structure, e.g., hairpin.
  • the present pharmaceutical composition for treating or preventing cancer may include either the double-stranded molecule (e.g., siRNA) or a vector expressing the double-stranded molecule in vivo.
  • the present invention provides pharmaceutical compositions for treating or preventing cancer that include a double-stranded molecule that inhibits the expression of the ADAMTS18 gene, or a vector expressing the double-stranded molecule in vivo.
  • the present invention also provides pharmaceutical compositions for inhibiting cancer cell proliferation, such composition including a double-stranded molecule which inhibits the expression of the ADAMTS18 gene, or a vector expressing the double-stranded molecule in vivo.
  • transfection-enhancing agent can be used for introducing the double-stranded molecule vector into the cell. FuGENE6 (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent. Therefore, the present pharmaceutical composition may further include such transfection-enhancing agents.
  • the present invention also provides the use of the double-stranded nucleic acid molecules of the present invention or vector encoding thereof in manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene.
  • the present invention relates to a use of double-stranded nucleic acid molecule that inhibits the expression of ADAMTS18 gene in a cell that over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 11 or 12 for manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene.
  • the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the ADAMTS18 gene.
  • the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene, wherein the method or process includes step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of ADAMTS18 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 11 or 12 as active ingredients.
  • the present invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene, wherein the method or process includes step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of ADAMTS18 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 11 or 12.
  • Antisense nucleic acids targeting the ADAMTS18 gene can be used to reduce the expression level of the gene that is up-regulated in cancerous cells including lung and esophageal cancer cells. Such antisense nucleic acids are useful for the treatment of cancer, in particular lung and esophageal cancer and thus are also encompassed by the present invention.
  • An antisense nucleic acid acts by binding to the nucleotide sequence of the ADAMTS18 gene, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the gene, promoting the degradation of the mRNAs, and/or inhibiting the expression of the protein encoded by the gene.
  • an antisense nucleic acid inhibits the ADAMTS18 protein to function in the cancerous cell.
  • the phrase “antisense nucleic acids” refers to nucleotides that specifically hybridize to a target sequence and includes not only nucleotides that are entirely complementary to the target sequence but also that include mismatches of one or more nucleotides.
  • the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least 70% or higher, preferably of at least 80% or higher, more preferably of at least 90% or higher, even more preferably of at least 95% or higher over a span of at least 15 continuous nucleotides of the ADAMTS18 gene or the complementary sequence thereof. Algorithms known in the art can be used to determine such homology.
  • Antisense nucleic acids of the present invention act on cells that produce proteins encoded by the ADAMTS18 gene by binding to the DNA or mRNA of the gene, inhibiting their transcription or translation, promoting the degradation of the mRNA, and inhibiting the expression of the protein, finally inhibiting the protein to function.
  • Antisense nucleic acids of the present invention can be made into an external preparation, such as a liniment or a poultice, by admixing it with a suitable base material which is inactive against the nucleic acids.
  • the antisense nucleic acids of the present invention can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such.
  • An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives of these. These can be prepared by following known methods.
  • the antisense nucleic acids of the present invention inhibit the expression of the ADAMTS18 gene and are useful for suppressing the biological activity of the protein.
  • expression-inhibitors including antisense nucleic acids of the present invention, are useful in that they can inhibit the biological activity of the ADAMTS18 protein.
  • the antisense nucleic acids of present invention also include modified oligonucleotides. For example, thioated oligonucleotides may be used to confer nuclease resistance to an oligonucleotide.
  • compositions including antibodies
  • the function of a gene product of the ADAMTS18 gene which is over-expressed in cancers, in particular lung and esophageal cancer can be inhibited by administering a compound that binds to or otherwise inhibits the function of the gene products.
  • An antibody against the ADAMTS18 polypeptide can be mentioned as such a compound and can be used as the active ingredient of a pharmaceutical composition for treating or preventing cancer.
  • the present invention relates to the use of antibodies against a protein encoded by the ADAMTS18 gene, or fragments of the antibodies.
  • antibody refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto. Molecules including the antigen that was used for synthesizing the antibody and molecules including the epitope of the antigen recognized by the antibody can be mentioned as closely related antigens thereto.
  • an antibody used in the present pharmaceutical compositions may be a fragment of an antibody or a modified antibody, so long as it binds to the protein encoded by the ADAMTS18 gene (e.g., an immunologically active fragment of anti- ADAMTS18 antibody).
  • the antibody fragment may be Fab, F(ab') 2 , Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston JS et al., Proc Natl Acad Sci USA 1988, 85:5879-83).
  • Such antibody fragments may be generated by treating an antibody with an enzyme, such as papain or pepsin.
  • a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co MS et al., J Immunol 1994, 152:2968-76; Better M et al., Methods Enzymol 1989, 178:476-96; Pluckthun A et al., Methods Enzymol 1989, 178:497-515; Lamoyi E, Methods Enzymol 1986, 121:652-63; Rousseaux J et al., Methods Enzymol 1986, 121:663-9; Bird RE et al., Trends Biotechnol 1991, 9:132-7).
  • An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present invention includes such modified antibodies.
  • the modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.
  • the antibody used for the present invention may be a chimeric antibody having a variable region derived from a non-human antibody against the ADAMTS18 polypeptide and a constant region derived from a human antibody, or a humanized antibody, including a complementarity determining region (CDR) derived from a non-human antibody, a frame work region (FR) and a constant region derived from a human antibody.
  • CDR complementarity determining region
  • FR frame work region
  • Such antibodies can be prepared by using known technologies. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see e.g., Verhoeyen et al., Science 1988, 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • Human antibodies including human variable regions in addition to human framework and constant regions can also be used.
  • Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom et al., J Mol Biol 1992, 227:381-8).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
  • Antibodies obtained as above may be purified to homogeneity.
  • the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins.
  • the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and D Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto.
  • a protein A column and protein G column can be used as the affinity column.
  • Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
  • Exemplary chromatography, with the exception of affinity includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)).
  • the chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
  • Cancer therapies directed at specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-tumor pharmaceuticals such as trastuzumab (Herceptin) for the treatment of advanced cancers, imatinib mesylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F et al., Clin Cancer Res 2001, 7:2958-70, Review; Slamon DJ et al., N Engl J Med 2001, 344:783-92; Rehwald U et al., Blood 2003, 101:420-4; Fang G et al., Blood 2000, 96:2246-53).
  • trastuzumab Herceptin
  • Imatinib mesylate for chronic myeloid leukemia
  • modulatory methods can be performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the methods involve administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid molecules as therapy to counteract aberrant expression of the differentially expressed genes or aberrant activity of their gene products.
  • Diseases and disorders characterized by increased (relative to a subject not suffering from the disease or disorder) expression levels or biological activities of genes and gene products, respectively, may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene.
  • therapeutics that antagonize activity can be administered therapeutically or prophylactically.
  • the dysfunctional antisense molecules are utilized to "knockout" endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 1989, 244: 1288
  • Increased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered).
  • Methods that are well known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
  • hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • Therapeutic methods of the present invention may include the step of administering an agent that modulates one or more of the activities of the ADAMTS18 gene products.
  • agent that modulate protein activity include, but are not limited to, nucleic acids, proteins, naturally occurring cognate ligands of such proteins, peptides, peptidomimetics, and other small molecule.
  • the present invention provides methods for treating or alleviating a symptom of cancer, or preventing cancer in a subject by decreasing the expression of the ADAMTS18 gene or the activity of the gene product.
  • the present method is particularly suited for treating or preventing lung and esophageal cancer.
  • Suitable therapeutics can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing cancers.
  • Such subjects can be identified by using standard clinical methods or by detecting an aberrant expression level ("up-regulation” or "over-expression") of the ADAMTS18 gene or aberrant activity of the gene product.
  • substances identified through the screening methods of the present invention may be used for treating or preventing cancer.
  • Methods well known to those skilled in the art may be used to administer the substances to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration.
  • the substances are encodable by a DNA
  • the DNA can be inserted into a vector for gene therapy and the vector can be administered to a patient to perform the therapy.
  • the dosage and methods for administration vary according to the body-weight, age, sex, symptom, condition of the patient to be treated and the administration method; however, one skilled in the art can routinely select suitable dosage and administration method.
  • the dose of a substance that binds to an ADAMTS18 polypeptide or regulates the activity of the polypeptide depends on the aforementioned various factors, the dose is generally about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult human (60 kg weight).
  • the agent When administering the agent parenterally, in the form of an injection to a normal adult human (60 kg weight), although there are some differences according to the patient, target organ, symptoms and methods for administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. In the case of other animals, the appropriate dosage amount may be routinely calculated by converting to 60 kg of body-weight.
  • compositions of the present invention may be used for treating or preventing cancer.
  • Methods well known to those skilled in the art may be used to administer the compositions to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration.
  • the compositions e.g., polypeptides and organic compounds
  • the dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day.
  • Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.
  • the dose employed will depend upon a number of factors, including the age, body weight and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity. In any event, appropriate and optimum dosages may be routinely calculated by those skilled in the art, taking into consideration the above-mentioned factors.
  • an antisense nucleic acid against the ADAMTS18 gene can be given to the patient by direct application onto the ailing site or by injection into a blood vessel so that it will reach the site of ailment.
  • the dosage of the antisense nucleic acid derivatives of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
  • the method of the present invention includes the step of administering the double-stranded molecule against the ADAMTS18 gene to a subject.
  • the preferred examples of the double-stranded molecule to be administered are described under the item of " V-2.
  • Double-stranded molecules and vectors encoding them It is understood that the double-stranded molecules of the present invention degrade the mRNA of the ADAMTS18 gene in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.
  • an effective amount of the double-stranded molecule of the present invention can readily determine an effective amount of the double-stranded molecule of the present invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic.
  • an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered. The precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
  • the present methods can be used to inhibit the growth or metastasis of cancer; for example lung cancer, especially lung cancer and esophageal cancer.
  • the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule.
  • the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer.
  • the double-stranded molecule of the present invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents).
  • the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector that expresses the double-stranded molecule.
  • Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • a preferred delivery reagent is a liposome.
  • Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule.
  • Liposomes suitable for use in the context of the present invention may be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
  • the liposomes encapsulating the present double-stranded molecule includes a ligand molecule that can deliver the liposome to the cancer site.
  • Ligands which bind to receptors prevalent in tumor or vascular endothelial cells such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.
  • the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure.
  • a liposome of the invention can include both opsonization-inhibition moieties and a ligand.
  • Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
  • an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
  • These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No.
  • Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
  • Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature.
  • target tissue characterized by such microvasculature defects for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53.
  • the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen.
  • liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
  • Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons.
  • Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • synthetic polymers such as polyacrylamide or poly N-vinyl pyr
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
  • the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques.
  • an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
  • Vectors expressing a double-stranded molecule of the present invention are discussed in the following item. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for delivering recombinant viral vectors, which express a double-stranded molecule of the invention, to an area of cancer in a patient are within the skill of the art.
  • the double-stranded molecule of the present invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites.
  • the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
  • Suitable enteral administration routes include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of the cancer.
  • the double-stranded molecule of the present invention can be administered in a single dose or in multiple doses.
  • the infusion can be a single sustained dose or can be delivered by multiple infusions.
  • Injection of the agent directly into the tissue is at or near the site of cancer preferred. Multiple injections of the agent into the tissue at or near the site of cancer are particularly preferred.
  • the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site.
  • the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days.
  • the double-stranded molecule is injected at or near the site of cancer once a day for seven days.
  • the effective amount of a double-stranded molecule administered to the subject can include the total amount of a double-stranded molecule administered over the entire dosage regimen.
  • a cancer overexpressing ADAMTS18 can be treated with at least one active ingredient selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof.
  • cancer to be treated examples include, but are not limited to, lung and esophageal cancer. Accordingly, prior to the administration of a double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of ADAMTS18 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ.
  • the present invention provides a method for treating a cancer (over)expressing ADAMTS18, which method may include the steps of: i) determining the expression level of ADAMTS18 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of ADAMTS18 with normal control; and iii) administrating at least one component selected from the group consisting of (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, to a subject with a cancer overexpressing ADAMTS18 compared with normal control.
  • the present invention also provides a pharmaceutical composition including at least one component selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, for use in administrating to a subject having a cancer overexpressing ADAMTS18.
  • the present invention further provides a method for identifying a subject to be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof, which method may include the step of determining an expression level of ADAMTS18 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with a double-stranded molecule of the present invention.
  • a subject to be treated by the present method is preferably a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the expression level of ADAMTS18 in cancer cells or tissues obtained from a subject is determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, hybridization methods (e.g., Northern hybridization), a chip or an array, probes, RT-PCR can be used to determine the transcription product level of ADAMTS18.
  • the translation product may be detected for the treatment of the present invention.
  • the quantity of observed protein SEQ ID NO: 2 may be determined.
  • the intensity of staining may be measured via immunohistochemical analysis using an antibody against the ADAMTS18 protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of ADAMTS18 gene.
  • Methods for detecting or measuring the ADAMTS18 polypeptide and/or polynucleotide encoding thereof can be exemplified as described above (I. Diagnosing cancer).
  • Double-stranded molecules and vectors encoding them an siRNA including either of the sequences of SEQ ID NOs: 11 or 12 was demonstrated to suppress cell growth or viability of cells expressing the ADAMTS18 gene. Therefore, double-stranded molecules including any of these sequences and vectors expressing the molecules are considered to serve as preferable pharmaceutics for treating or preventing diseases which involve the proliferation of ADAMTS18 gene expressing cells, for example, cancer, particularly lung and esophageal cancer.
  • the present invention provides double-stranded molecules including the target sequence selected from the group consisting of SEQ ID NOs: 11 and 12 and vectors expressing the molecules.
  • the present invention provides a double-stranded molecule, when introduced into a cell expressing the ADAMTS18 gene, inhibits expression of the gene, wherein the double-stranded molecule includes a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 and 12 as a target sequence, and the antisense strand includes a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  • the present invention provides a double-stranded molecule, when introduced into a cell expressing an ADAMTS18 gene, inhibits expression of the gene, wherein the double-stranded molecule has a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 11 and 12, and the antisense strand has a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  • the target sequence for the ADAMTS18 gene included in the sense strand may consist of a sequence of a portion of SEQ ID NO: 1 that is less than about 500, 400, 300, 200, 100, 75, 50 or 25 contiguous nucleotides.
  • the target sequence may be from about 19 to about 25 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1.
  • suitable target sequences include the nucleotide sequences selected from the group consisting of SEQ ID NOs: 11 and 12.
  • the double-stranded molecule of the present invention may be composed of two polynucleotide constructs, i.e., a polynucleotide including the sense strand and a polynucleotide including the antisense strand.
  • the molecule may be composed of one polynucleotide construct; i.e., a polynucleotide including both the sense strand and the antisense strand, wherein the sense and antisense strands are linked via a single-stranded polynucleotide which enables hybridization of the target sequences within the sense and antisense strands by forming a hairpin structure.
  • the single-stranded polynucleotide may also be referred to as "loop sequence” or “single-strand".
  • the single-stranded polynucleotide linking the sense and antisense strands may consist of 3 to 23 nucleotides. See under the item of "V-2.
  • the double-stranded molecules of the present invention may contain one or more modified nucleotides and/or non-phosphodiester linkages.
  • Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule.
  • the skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; WO2005/045037).
  • modifications can be used to provide improved resistance to degradation or improved uptake.
  • modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base” nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).
  • modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule.
  • Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).
  • modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976).
  • an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine.
  • an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine.
  • the double-stranded molecule is a double-stranded molecule with a 3' overhang
  • the 3'- terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200).
  • published documents such as US20060234970 are available.
  • the present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
  • the double-stranded molecules of the invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA.
  • RNA e.g., dsD/R-NA or shD/R-NA.
  • a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability.
  • RNA i.e., a hybrid type double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule including both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule.
  • the hybrid of a DNA strand and an RNA strand may be the hybrid in which either the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA.
  • the chimera type double-stranded molecule may be either the molecule that both of the sense and antisense strands are composed of DNA and RNA, or the molecule that any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression.
  • an upstream partial region i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands
  • the upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand.
  • a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA.
  • the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
  • sense strand 5'-[---DNA---]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(---RNA---)-5' :antisense strand
  • the upstream partial region preferably is a domain consisting of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules.
  • preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
  • the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA).
  • shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence.
  • the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the present invention provides vectors including each of a combination of polynucleotide having a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid includes nucleotide sequence of SEQ ID NOs: 11 or 12, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the ADAMTS18, inhibit expression of said gene.
  • the sense strand of the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1). More preferably, the combination of polynucleotide includes a single nucleotide transcript having the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
  • the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is a nucleotide sequence including SEQ ID NO: 11 or 12; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to [A].
  • Vectors of the present invention can be produced, for example, by cloning ADAMTS18 sequence into an expression vector so that regulatory sequences are operatively-linked to ADAMTS18 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5).
  • RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • a first promoter e.g., a promoter sequence flanking to the 3' end of the cloned DNA
  • RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene.
  • two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and anti-sense strands and then forming a double-stranded molecule construct.
  • the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
  • the vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).
  • the vectors of the present invention include, for example, viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell.
  • Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • a wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
  • the 15 human lung-cancer cell lines used in this example included: five adenocarcinomas (ADCs; NCI-H1781, NCI-H1373, LC319, A549 and PC-14), five squamous-cell carcinomas (SCCs; SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170 and LU61), one large-cell carcinoma (LCC; LX1), and four small-cell lung cancers (SCLCs; SBC-3, SBC-5, DMS114 and DMS273).
  • ADCs adenocarcinomas
  • SCCs squamous-cell carcinomas
  • LCC large-cell carcinoma
  • SCLCs small-cell lung cancers
  • the 10 human esophageal carcinoma cell lines used in this study were as follows; 9 SCC cell lines (TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, and TE10) and one ADC cell line (TE7) (Nishihira T, et al. J Cancer Res Clin Oncol 1993;119:441-9.). All cells were grown in monolayer in appropriate media supplemented with 10% fetal calf serum (FCS) and were maintained at 37 degrees C in humidified air with 5% CO 2 . Human small airway epithelial cells (SAEC) used as a normal control were grown in optimized medium (SAGM) from Cambrex Bio Science Inc.
  • FCS fetal calf serum
  • RNA samples were reversely transcribed to single-stranded cDNAs using random primer (Roche Diagnostics) and Superscript II (Invitrogen).
  • Semiquantitative RT-PCR experiments were carried out with the following sets of synthesized primers specific for human epithelial cell transforming sequence 2 (ADAMTS18) or with beta-actin (ACTB)-specific primers as an internal control: ADAMTS18, 5'-GGATTAGCCAGCTCAGCATA-3' (SEQ ID NO: 3) and 5'-CTGTTTTTCAGAAGGCAACG-3' (SEQ ID NO: 4); ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3' (SEQ ID NO: 5) and 5'-CAAGTCAGTGTACAGGTAAGC-3' (SEQ ID NO: 6). PCR reactions were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification.
  • RNA interference assay To evaluate the biological functions of ADAMTS18 in cancer cells, a short-hairpin RNA against the target genes were used.
  • the target sequences of the synthetic oligonucleotides for RNAi were as follows: control 1 (enhanced green fluorescent protein gene (EGFP), a mutant of Aequorea victoria GFP), 5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID NO: 9); control 2 (Luciferase (LUC): Photinus pyralis luciferase gene), 5'-CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 10); si-ADAMTS18-#1, 5'-GCCAGUAUCUCAAGAAAUU-3' (SEQ ID NO: 11); si-ADAMTS18-#2, and 5'-GGGCACAACUUUGGUAUGA-3' (SEQ ID NO: 12).
  • DMS114 and TE4 cells were plated onto 10-cm dishes (1.5x 10 6 cells per dish), and transfected with either of the siRNA oligonucleotides (100nM), using 24 microliter of Lipofectamine 2000 (Invitrogen), according to the manufacturers' instructions. After 7 days of incubation, these cells were stained by Giemsa solution to assess colony formation, and cell numbers were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • ADAMTS18 expression plasmids Preparation of ADAMTS18 expression plasmids. To investigate the biological function of ADAMTS18, p3XFLAG-tagged (C-terminal) plasmids expressing full-length fragments of ADAMTS18 were prepared.
  • the membrane was incubated with ANTI-FLAG, antibody produced in rabbit (SIGMA) for 1 hour at room temperature. Immunoreactive proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 hour at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-sciences). When cell culture media was applied for western-blotting, supernatant was collected after centrifugation for 10-15 minutes at 1,000X g, 4 degrees C. Then, supernatant were concentrated by using Amicon Ultra-15 Centrifugal Filter Devices (MILLIPORE), according to the manufacturers' instructions.
  • MILLIPORE Amicon Ultra-15 Centrifugal Filter Devices
  • COS-7 and A549 cells transfected either with p3XFLAG-tagged (C-terminal) plasmids expressing ADAMTS18 or with mock plasmids were seeded on to 6-well microtiter plates (1 x 10 4 cells/well). After 7 days of incubation, these cells were evaluated using Cell Counting Kits as directed by the supplier (Wako).
  • N-glycosidase assay COS-7 cells transfected either with p3XFLAG-tagged (C-terminal) plasmids expressing ADAMTS18 or with mock plasmids were harvested as described above, and each samples were mixed with N-Glycosidase F (CALBIOCHEM). After incubation at 37 degrees C for 16 hours, Western-blotting analysis was performed.
  • each specimen was mounted with Vectashield (Vector Laboratories) containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS, Leica Microsystems).
  • DAPI 4',6-diamidino-2-phenylindole dihydrochloride
  • DMEM fetal calf serum
  • Matrix metalloproteinase assay Two types of cells were prepared; COS-7 cells transfected either with p3XFLAG-tagged (C-terminal) plasmids expressing ADAMTS18 or with mock plasmids were grown to near confluence in DMEM containing 10% FCS, whereas DMS114 cells transfected either with si-ADAMTS18-#1 or with control 1 (EGFP) were incubated for 48 hours. Then, supernatant of cell culture media were collected and centrifuged for 10-15 minutes at 1,000X g, at 4 degrees C.
  • MMP Matrix Metalloproteinase
  • SensoLyte 570 Generic Assay Kit AmaSpec
  • the MMP containing-samples were incubated with 1 mM 4-aminophenyl mercuric acetate for 2 hours to activate MMP.
  • the samples and generic MMP substrate (containing substrates of MMPs-1, 2, 7, 8, 9, 10, 13 and 14) were mixed to start enzymatic reaction and incubated at room temperature for 30 minutes. Fluorescence intensity was measured by Microplate reader.
  • ADAMTS18 was considered to be a good molecular candidate for further analyses. Its overexpression was confirmed by semiquantitative RT-PCR experiments in 7 of 15 lung cancer tissues, in 2 of 15 lung-cancer cell lines, in 5 of 10 ESCC tissues, and in 1 of 10 ESCC cell lines examined (Figs. 1A and 1B). In 5 ESCC patients, the ADAMTS18 expression between ESCC tissues and their normal esophagus tissues was also compared (Figs. 1C). Northern blot analysis using an ADAMTS18 cDNA as a probe identified a transcript of 6.0-kb only in placenta among 23 normal human tissues examined (Fig. 1D).
  • ADAMTS18 Inhibition of growth of cancer cells by small interfering RNA for ADAMTS18.
  • synthetic oligonucleotide siRNAs against ADAMTS18 was used and transfected into DMS114 and TE4 cells that endogenously expressed high levels of ADAMTS18.
  • the ADAMTS18-mRNA levels in cells transfected with si-ADAMTS18-#1 or -#2 were significantly decreased in comparison with cells transfected with either control siRNAs (Fig. 2, top panels).
  • MTT assays and colony-formation assays revealed a drastic reduction in the number of cells transfected with si-ADAMTS18-#1 or -#2 (Fig. 2, middle and bottom panels).
  • plasmids designed to express ADAMTS18 was prepared and transfected into COS-7 cells. After confirmation of ADAMTS18 expression by western-blotting (Fig. 3A, left panels), MTT assays was carried out, and it was found that growth of the COS-7-ADAMTS18 cells was promoted at a significant degree in comparison to the COS-7 cells transfected with the mock vector (Fig. 3A, right panel).
  • A549 cells were transfected as same as COS-7 cells, and it revealed that exogenous ADAMTS18 promoted cell growth of A549 (Figs. 3B).
  • ADAMTS18 Post-translational modification of ADAMTS18.
  • N-glycosidase assay was performed. After incubation with N-glycosidase, western-blot analysis revealed that the band of exogenous ADAMTS18 protein was shifted lower (Fig. 4A). The results suggested that ADAMTS18 could be N-glycosylated after translation.
  • ADAMTS18 secretion of exogenous ADAMTS18 protein into culture medium.
  • ADAMTS18 was supposed to be a secreted protein, so the secretion of ADAMTS18 protein into culture medium was evaluated by using COS-7 cells, which were not or very lower expressed ADAMTS18 (Fig. 4C), overexpressed ADAMTS18.
  • COS-7 cells which were not or very lower expressed ADAMTS18 (Fig. 4C)
  • ADAMTS18 overexpressed ADAMTS18.
  • culture medium was changed. Then, at 72-hour after transfection, culture medium was harvested. After centrifugation, culture medium was concentrated and applied for western-blotting analysis. Exogenous ADAMTS18 protein was appeared to be secreted into culture medium (Fig. 4D).
  • ADAMTS18 Activation of mammalian cellular invasion by ADAMTS18. Because of ADAMTS18 contains metalloproteinase domain, which digests ECM (extra cellular matrix), a possible role of ADAMTS18 in cellular invasion was examined by Matrigel assays using a mammalian cells (COS-7). Transfection of ADAMTS18 cDNA into the cells significantly enhanced their invasive activity through Matrigel, compared to cells transfected with mock vector (Fig. 5A). And cell growth assays, performed at the same time point with Matrigel assays, revealed no significant differences between them (Fig. 5B). This result independently suggested that ADAMTS18 could contribute to the highly malignant phenotype of cancer cells.
  • COS-7 mammalian cells
  • MMP activity of ADAMTS18 was evaluated by MMP assay.
  • COS-7 cells which had been transfected with ADAMTS18-expressing plasmids significantly enhanced MMP activity, compared to cells transfected with mock vector (Fig. 5C).
  • lung cancer cells which had been transfected with si-ADAMTS18-#1, significantly showed decreased MMP activity in comparison with cells transfected with control siRNA (EGFP) (Fig. 5D).
  • the ADAM family of proteins an MMP-related metalloproteinase family, are multifunctional proteins involved in the proteolytic processing of other transmembrane proteins, cell adhesion and cell signaling events (Mochizuki S and Okada Y. Cancer Sci 2007;98:621-8).
  • Many transmembrane proteins are processed by one or several proteolytic steps to the biologically active configuration. Examples include growth factors such as EGF, HB-EGF and TGF-alpha, and cytokines such as TNF-alpha, all of which are synthesized as precursors.
  • EGF EGF
  • HB-EGF HB-EGF
  • TGF-alpha cytokines
  • cytokines such as TNF-alpha
  • TNF-alpha receptor-I TNF-alpha receptor-II
  • CD44 L-selectin
  • Erb4/HER4 Erb4/HER4.
  • the soluble, released ectodomains of the receptors may be part of the down-modulation in response to ligand activation, or they may have a function of their own. It has become clear over the past few years that ADAMs play a major role in these processes.
  • ADAMs matrix metalloproteinases
  • ADAMTSs a disintegrin and metalloproteinase with thrombospondin motifs
  • ADAMTS18 was characterized as a novel oncoprotein involved in lung and esophageal cancers.
  • the importance of ADAMTS18 was evaluated for cancer cell growth and/or survival.
  • the treatment of two cancer cells with specific siRNA for suppression of ADAMTS18 expression resulted in inhibition of cancer-cell growth. Additional evidences supporting the significance of ADAMTS18 in carcinogenesis were also obtained.
  • the induction of exogenous ADAMTS18 into mammalian COS-7 cells and A549, lung cancer cells resulted in the significant promotion of cell growth.
  • induction of exogenous ADAMTS18 into COS-7 cells activated cellular invasion.
  • ADAMTS18 was secreted into culture medium and might have MMP activity.
  • ADAMs have been shown to promote cancer initiation and progression, one can reasonable predict that blocking their actions would slow or prevent tumor progression.
  • a number of selective synthetic inhibitors against a small number of ADAMs have been described (Duffy MJ, McKiernan E, O'Donovan N, and McGowan PM. Clin Cancer Res 2009;15:1140-4).
  • ADAMTS18 is properly classified as one of typical cancer-placenta antigens, selective inhibition of ADAMTS18 enzymatic activity by small molecule compounds constitutes a promising therapeutic strategy that is expected to have a powerful biological activity against cancer with a minimal risk of adverse events.
  • the data herein enable the design of new anti-cancer drugs to specifically target the oncogenic activity of ADAMTS18 for treatment of cancer patients.
  • ADAMTS18 was identified as a novel potential therapeutic target for the patients with lung and esophageal cancers.
  • the gene-expression analysis of cancers described herein using the genome-wide cDNA microarray has identified specific genes as a target for cancer prevention and therapy. Based on the differentially expression of ADAMTS18 gene, the present invention provides a molecular diagnostic marker for diagnosing or detecting cancer, in particular, lung and esophageal cancer.
  • the data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of a molecular target for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
  • ADAMTS18 a novel double-stranded molecules find real-world utility as anti-cancer pharmaceuticals.
  • the expression of the ADAMTS18 gene is markedly elevated in cancer, specifically lung cancer and esophageal cancer, as compared to normal organs. Accordingly, this gene finds convenient, real-world utility as a diagnostic marker for cancer, in particular, lung cancer and esophageal cancer, and the proteins encoded thereby find utility in diagnostic assays for cancer.
  • the methods described herein find utility in diagnosis of cancer, including lung cancer and esophageal cancer.
  • the present invention provides new therapeutic approaches for treating cancer including lung cancer and esophageal cancer.
  • the ADAMTS18 gene finds real-world utility as a useful target for the development of anti-cancer pharmaceuticals. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention provides methods for detecting and diagnosing cancer, such methods involving the determination of the expression level of the ADAMTS18 gene. This gene was discovered to discriminate cancer cells from normal cells. Furthermore, the present invention provides methods of screening for therapeutic agents useful in the treatment of cancer and methods for treating cancer. Moreover, the present invention provides double-stranded molecules targeting the ADAMTS18, all of which are suggested to be useful in the treatment of cancer.

Description

LUNG AND ESOPHAGEAL CANCER RELATED GENE ADAMTS18
The present invention relates to methods for detecting and diagnosing cancer as well as methods for treating and preventing cancer.
Priority
The present application claims the benefit of U.S. Provisional Application No. US 61/275,039, filed on August 24, 2009, the entire contents of which are incorporated by reference herein.
BACKGROUND ART
Primary lung cancer is the leading cause of cancer deaths in most countries (NPL 1), and esophageal squamous cell carcinoma (ESCC) is one of the most common fatal malignancies of the digestive tract (NPL 2). Despite improvements in surgical techniques and adjuvant chemoradiotherapy, patients with advanced lung or esophageal cancer often suffer fatal disease progression (NPLs 1-2). Therefore, it is extremely important to understand the biology of these two major thoracic cancers and to introduce more effective treatments to improve the survival of patients (NPL 3).
The concept of specific molecular targeting has been applied to the development of innovative cancer treatment strategies. At present, two main approaches are available in clinical practice: therapeutic monoclonal antibodies and small-molecule agents (NPL 4). To date, a number of targeted therapies such as bevacizumab, cetuximab, erlotinib, gefitinib, sorafenib, and sunitinib have been investigated in phase II and phase III trials for the treatment of advanced non-small-cell lung cancer (NSCLC; NPLs 4-8). The addition of therapeutic antibodies against proangiogenic protein vascular endothelial growth factor (bevacizumab) or epidermal growth factor receptor (cetuximab) to conventional chemotherapy has afforded significant survival benefits to patients with NSCLC (NPLs 4-5). Two small-molecule epidermal growth factor receptor tyrosine kinase inhibitors, erlotinib and gefitinib, have been shown to be effective for a subset of advanced NSCLC patients (NPLs 6-7). Phase II studies done with two oral multi-targeted receptor tyrosine kinase inhibitors, sorafenib (inhibitor for c-RAF, b-RAF, vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor h, and KIT) and sunitinib (inhibitor for platelet-derived growth factor receptor, KIT, FLT3, and vascular endothelial growth factor receptor) suggested their efficacy in the treatment of advanced NSCLCs (NPL 8).
However, issues of toxicity limit these treatment regimens to selected patients, and even if all kinds of available treatments are applied, the proportion of patients exhibiting a positive response is still very limited (NPL 4-8). To isolate potential molecular targets for diagnosis, treatment, and/or prevention of lung and esophageal carcinomas, a genome-wide analysis of gene expression profiles of cancer cells from 101 lung-cancer and 19 ESCC patients was previously performed by means of a cDNA microarray consisting of 27,648 genes or expressed sequence tags (NPLs 3, 9-14). To verify the biological and clinicopathologic significance of the respective gene products, a screening system by a combination of the tumor tissue microarray analysis of clinical lung and esophageal cancer materials with RNA interference technique (NPLs 15-33).
This screening system resulted in the identification of ADAMTS18 as an agent up-regulated in small cell lung cancer cells (WO2007/013665). There are two subgroups in the ADAM family: membrane-anchored ADAM and secreted-type ADAMTS. ADAM members are composed of common domains including propeptide, metalloproteinase, disintegrin, cysteine-rich, EGF-like, transmembrane and cytoplasmic domains, whereas ADAMTS members contain thrombospondin motifs, cysteine-rich and spacer domains in addition to propeptide, metalloproteinase and disintegrin domains (NPL 34). Although ADAMTSs are soluble proteins, many of them appear to bind the extracellular matrix through their thrombospondin motifs or their spacer region (NPL 35). With the exception of ADAMTS10 and -12, ADAMTSs are regulated through a proteolytic processing occurring at the furin-like recognition site located between the pro- and catalytic domains (NPLs 35-36). Dysregulation of the production of several ADAMs and ADAMTSs has been documented in lung cancers (NPLs 16, 37-45) and other cancers (NPL 37), such as brain tumors, prostate cancer, liver carcinoma, breast cancer, etc. In spite of these reports, the significance of activation of ADAMTS18 in human cancer progression and its clinical potential as a therapeutic target have not yet been fully described.
[PTL 1] PCT/JP2006/315254
[NPL 1] Ahmedin J, Rebecca S, Elizabeth W, et al. Cancer statistics, 2007.CA Cancer J Clin 2007;57:43-66
[NPL 2] Shimada H, Nabeya Y, Ochiai T, et al. Surgery 2003;133:486-94
[NPL 3] Daigo Y, Nakamura Y. Gen Thorac Cardiovasc Surg 2008;56:43-53
[NPL 4] Thatcher N. Lung Cancer 2007;57 Suppl 2:S18-23
[NPL 5] Sandler A, Gray R, Perry MC, et al. N Engl J Med 2006;355:2542-50
[NPL 6] Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. N Engl J Med 2005;353:123-32
[NPL 7] Thatcher N, Chang A, Parikh P, et al. Lancet 2005;366:1527-37
[NPL 8]Cesare G, Paolo M, Filomena G, et al. Oncologist 2007;12:191-200
[NPL 9]Kikuchi T, Daigo Y, Katagiri T, et al. Oncogene 2003;22:2192-205
[NPL 10] Kakiuchi S, Daigo Y, Tsunoda T, et al. Mol Cancer Res 2003;1:485-99
[NPL 11] Kakiuchi S, Daigo Y, Ishikawa N, et al. Hum Mol Genet 2004;13:3029-43
[NPL 12] Kikuchi T, Daigo Y, Ishikawa N, et al. Int J Oncol 2006; 28:799-805
[NPL 13] Taniwaki M, Daigo Y, Ishikawa N, et al. Int J Oncol 2006;29:567-75
[NPL 14] Yamabuki T, Daigo Y, Kato T, et al. Int J Oncol 2006;28:1375-84
[NPL 15] Suzuki C, Daigo Y, Kikuchi T, et al. Cancer Res 2003;63:7038-41
[NPL 16] Ishikawa N, Daigo Y, Yasui W, et al. Clin Cancer Res 2004;10:8363-70
[NPL 17] Kato T, Daigo Y, Hayama S, et al. Cancer Res 2005;65:5638-46
[NPL 18] Furukawa C, Daigo Y, Ishikawa N, et al. Cancer Res 2005;65:7102-10
[NPL 19] Ishikawa N, Daigo Y, Takano A, et al. Cancer Res 2005; 65:9176-84
[NPL 20] Suzuki C, Daigo Y, Ishikawa N, et al. Cancer Res 2005;65:11314-25
[NPL 21] Ishikawa N, Daigo Y, Takano A, et al. Cancer Sci 2006;97:737-45
[NPL 22] Takahashi K, Furukawa C, Takano A, et al. Cancer Res 2006;66:9408-19
[NPL 23] Hayama S, Daigo Y, Kato T, et al. Cancer Res 2006;66:10339-48
[NPL 24] Kato T, Hayama S, Yamabuki Y, et al. Clin Cancer Res 2007;13:434-42
[NPL 25] Suzuki C, Takahashi K, Hayama S, et al. Mol Cancer Ther 2007;6:542-51
[NPL 26] Yamabuki T, Takano A, Hayama S, et al. Cancer Res 2007;67:2517-25
[NPL 27] Hayama S, Daigo Y, Yamabuki T, et al. Cancer Res 2007;67:4113-22
[NPL 28] Kato T, Sato N, Hayama S, et al. Cancer Res 2007;67:8544-53
[NPL 29] Taniwaki M, Takano A, Ishikawa N, et al. Clin Cancer Res 2007;13:6624-31
[NPL 30] Ishikawa N, Takano A, Yasui W, et al. Cancer Res 2007;67:11601-11
[NPL 31] Mano Y, Takahashi K, Ishikawa N, et al. Cancer Sci 2007;98:1902-13
[NPL 32] Suda T, Tsunoda T, Daigo Y, et al. Cancer Sci 2007;98:1803-8
[NPL 33] Kato T, Sato N, Takano A, et al. Clin Cancer Res 2008;in press
[NPL 34] Mochizuki S and Okada Y. Cancer Sci 2007;98:621-8
[NPL 35] Porter S, Clark IM, Kevorkian L, and Edwards DR. Biochem J 2005;386:15-27
[NPL 36] Cal S, Obaya AJ, Llamazares M, Garabaya C, et al. Gene 2002;283:49-62
[NPL 37] Rocks N, Paulissen G, El Hour M, et al. Biochimie 2008;90:369-79
[NPL 38] Lemjabbar H, Li D, Gallup M, Sidhu S, Drori E, Basbaum C. J Biol Chem 2003;278:26202-7
[NPL 39] Zhou BB, Peyton M, He B, et al. Cancer Cell 2006;10:39-50
[NPL 40] Shintani Y, Higashiyama S, Ohta M, et al. Cancer Res 2004;64:4190-6
[NPL 41] Rocks N, Paulissen G, Quesada CF, et al. Br J Cancer 2006;94:724-30
[NPL 42] Mochizuki S, Shimoda M, Shiomi T, et al. Biochem Biophys Res Commun 2004;315:79-84
[NPL 43] Ohtsuka T, Shiomi T, Shimoda M, et al. Int J Cancer 2006;118:263-73.
[NPL 44] Schutz A, Hartig W, Wobus M, et al. Virchows Arch 2005;446:421-9
[NPL 45] Dunn JR, Panutsopulos D, Shaw MW, et al. Br J Cancer 2004;91:1149-54
The present invention relates to the discovery of a specific expression pattern of the ADAMTS18 gene in cancerous cells.
Through the present invention, the ADAMTS18 gene was revealed to be frequently up-regulated in human tumors, in particular, lung and esophageal tumors. Moreover, since the suppression of the ADAMTS18 gene by small interfering RNA (siRNA) resulted in growth inhibition and/or cell death of lung and esophageal cancer cells, this gene may serve as a novel therapeutic target for human lung and esophageal cancers.
The ADAMTS18 gene identified herein, as well as its transcription and translation products, finds diagnostic utility as a marker for lung and esophageal cancer and as an oncogene target, the expression and/or activity of which may be altered to treat or alleviate a symptom of cancer. Similarly, by detecting changes in the expression of the ADAMTS18 gene and/or the activity of the ADAMTS18 protein that arise from exposure to a test substance, various agents for treating or preventing cancer can be identified.
Accordingly, it is an object of the present invention to provide a method for diagnosing or determining a predisposition to lung or esophageal cancer in a subject by determining the expression level of the ADAMTS18 gene in a subject-derived biological sample, such as tissue sample. An increase in the level of expression of the gene as compared to a normal control level indicates that the subject suffers from or is at risk of developing lung or esophageal cancer.
It is another object of the present invention to provide a kit that includes at least one reagent for detecting a transcription or translation product of the ADAMTS18 gene.
It is yet another object of the present invention to provide a reagent for diagnosis or detection of cancer that includes a nucleic acid that binds to a transcriptional product of the ADAMTS18 gene, or an antibody that binds to a translational product of the ADAMTS18 gene.
It is yet another object of the present invention to provide use of a nucleic acid that binds to a transcriptional product of the ADAMTS18 gene, or an antibody that binds to a translational product of the ADAMTS18 gene for the manufacture of a reagent for diagnosis or detection of cancer.
It is yet another object of the present invention to provide methods for identifying substances that bind the ADAMTS18 protein, by contacting the ADAMTS18 protein with a test substance and detecting the binding between the ADAMTS18 protein and the test substance. Test substances that bind the ADAMTS18 protein may be used to reduce symptoms of lung or esophageal cancer, or treating and/or preventing lung cancer or esophageal cancer.
It is yet another object of the present invention to provide methods for identifying substances that inhibit the biological activity of the ADAMTS18 protein, by contacting the ADAMTS18 protein with a test substance and detecting the biological activity of the ADAMTS18 protein. The biological activity of the ADAMTS18 protein to be detected is preferably cell proliferative activity (cell proliferation enhancing activity), N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity. A decrease in the biological activity of the ADAMTS18 protein as compared to a control level in the absence of the test substance indicates that the test substance may be used to reduce symptoms of lung or esophageal cancer, or treating and/or preventing lung cancer or esophageal cancer.
It is yet another object of the present invention to provide methods for identifying substances that inhibit the expression of the ADAMTS18 gene, by contacting a test cell expressing the ADAMTS18 gene with a test substance and determining the expression level of the ADAMTS18 gene. The test cell may be an epithelial cell, such as cancerous epithelial cell. A decrease in the expression level of the gene as compared to a control level in the absence of the test substance indicates that the test substance may be used to reduce symptoms of lung and esophageal cancer or treating and/or preventing lung cancer or esophageal cancer.
The present invention confirms the inhibitory effect of siRNAs for the ADAMTS18 gene. In particular, the inhibition of cell proliferation of cancer cells by the siRNAs is demonstrated in the Examples section. Thus, the data herein support the utility of the ADAMTS18 gene as a preferred therapeutic target for lung and esophageal cancer and the utility of double-stranded molecules against the ADAMTS18 gene as cancer therapeutics.
Accordingly, it is another object of the present invention is to provide a double-stranded molecule that inhibits the expression of the ADAMTS18 gene as well as siRNAs against the ADAMTS18 gene, and a vector encoding the double-stranded molecule. A double-stranded molecule of the present invention inhibits expression of the gene when introduced into a cell expressing an ADAMTS18 gene and is composed of a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 and 12 as a target sequence, and the antisense strand has a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
It is yet another object of the present invention is to provide a method for treating and/or preventing cancer in a subject, or inhibiting cancer cell growth. Therapeutic methods of the present invention include the step of administering an antisense composition to the subject. In the context of the present invention, the antisense composition reduces the expression of the ADAMTS18 gene. For example, the antisense compositions may contain a nucleotide that is complementary to the ADAMTS18 gene sequence. Alternatively, the present methods may include the step of administering an siRNA or double-stranded molecule composition to the subject. In the context of the present invention, the siRNA or double-stranded molecule composition reduces the expression of the ADAMTS18 gene. In yet another method, the treatment and/or prevention of lung and esophageal cancer in a subject may be carried out by administering a ribozyme composition to the subject. In the context of the present invention, the nucleic acid-specific ribozyme composition reduces the expression of the ADAMTS18 gene.
It is yet another object of the present invention is to provide a pharmaceutical composition formulated for use in treating and/or preventing cancer, or inhibiting cancer cell growth. A pharmaceutical composition of the present invention includes an antisense nucleotide or double-stranded molecule (e.g., siRNA) against ADAMTS18 gene that inhibits the expression of the ADAMTS18 gene.
It is yet another object of the present invention is to provide for the use of an antisense nucleotide or double-stranded molecule (e.g., siRNA) against ADAMTS18 gene, particularly in the context of cancer therapy and prevention, more particularly the treatment and/or prevention of lung and esophageal cancer.
More specifically, the present invention provides the following [1] to [26]:
[1] A method for detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of an ADAMTS18 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates that said subject suffers from or is at a risk of developing cancer, wherein said expression level is determined by any method selected from a group consisting of:
(a) detecting mRNA of an ADAMTS18 gene;
(b) detecting a protein encoded by an ADAMTS18 gene; and
(c) detecting a biological activity of a protein encoded by an ADAMTS18 gene,
[2] The method of [1], wherein said expression level is at least 10% greater than the normal control level,
[3] The method of [1] or [2], wherein the biological activity is cell proliferative activity, N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity,
[4] A method of screening a candidate substance for treating or preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof;
(b) detecting binding between the polypeptide or fragment and the test substance; and
(c) selecting the test substance that binds to the polypeptide or fragment as a candidate substance for treating or preventing cancer,
[5] A method of screening a candidate substance for treating or preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof;
(b) detecting a biological activity of the polypeptide or fragment;
(c) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the substance; and
(d) selecting the test substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer,
[6] The method of [5], wherein the biological activity is cell proliferative activity, N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity,
[7] A method of screening a candidate substance for treating or preventing cancer, which comprises steps of:
(a) contacting a test substance with a cell expressing an ADAMTS18 gene;
(b) detecting expression level of the ADAMTS18 gene;
(c) comparing the expression level with the expression level detected in the absence of the test substance; and
(d) selecting the test substance that reduces the expression level as a candidate substance for treating or preventing cancer,
[8] A method of screening a candidate substance for treating or preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with a cell introduced with a vector that comprises a transcriptional regulatory region of an ADAMTS18 gene and a reporter gene expressed under control of the transcriptional regulatory region;
(b) measuring an expression level or activity of said reporter gene;
(c) comparing the expression level or activity with the expression level or activity detected in the absence of the test substance; and
(d) selecting the test substance that reduces the expression level or activity as a candidate substance for treating or preventing cancer,
[9] A double-stranded molecule, when introduced into a cell expressing an ADAMTS18 gene, inhibits expression of the gene, wherein the double-stranded molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 11 and 12, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule,
[10] The double-stranded molecule of [9], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length,
[11] The double-stranded molecule of [9] or [10], wherein said double-stranded molecule is a single polynucleotide construct comprising the sense strand and the antisense strand linked via a single-strand,
[12] The double-stranded molecule of [11], which has a general formula 5'-[A]-[B]-[A']-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 11 and 12, [B] is a single-strand and consists of 3 to 23 nucleotides, and [A'] is an antisense strand comprising a nucleotide sequence complementary to the target sequence selected from SEQ ID NO: 11 and 12,
[13] A vector encoding the double-stranded molecule of any one of [9] to [12],
[14] Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises a nucleotide sequence corresponding to SEQ ID NO: 11 or 12, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing ADAMTS18 gene, inhibit the cell proliferation,
[15] A method of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against an ADAMTS18 gene or a vector encoding said double-stranded molecule, wherein the double-stranded molecule inhibits the expression of the ADAMTS18 gene,
[16] The method of [15], wherein the double-stranded molecule is that of any one of [9] to [12],
[17] The method of [15], wherein the vector is that of [13] or [14],
[18] The method of any one of [1] to [8], and [15] to [17], wherein the cancer is selected from the group consisting of lung cancer and esophagus cancer,
[19] A composition for treating or preventing cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against an ADAMTS18 gene or a vector comprising said double-stranded molecule, wherein the double-stranded molecule inhibits the expression of the ADAMTS18 gene, and a pharmaceutically acceptable carrier,
[20] The composition of [19], wherein the double-stranded molecule is that of any one of [9] to [12],
[21] The composition of [19], wherein the vector is that of [13] or [14],
[22] The composition of any one of [19] to [21], wherein the cancer is selected from the group consisting of lung cancer and esophagus cancer,
[23] A kit for diagnosing or detecting cancer, comprising a reagent for detecting a transcription or translation product of an ADAMTS18 gene,
[24] The kit of [23], wherein the reagent comprises a nucleic acid that binds to a transcription product of ADAMTS18 gene or an antibody that binds to a translation product of an ADAMTS18 gene,
[25] A reagent for diagnosing or detecting cancer, comprising a nucleic acid that binds to a transcription product of ADAMTS18 gene or an antibody that binds to a translation product of an ADAMTS18 gene, and
[26] The kit of [23] or [24], or the reagent of [25], wherein the cancer is selected from the group consisting of lung cancer and esophagus cancer.
One advantage of the methods described herein is that the disease is identified prior to detection of overt clinical symptoms of lung and esophageal cancer. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding objects can be viewed in the alternative with respect to any one aspect of this invention. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention.
Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the figures and the detailed description of the present invention and its preferred embodiments that follows:
Fig. 1 depicts the expression of ADAMTS18 in lung and esophageal cancers and normal tissues: Part A depicts the expression of ADAMTS18 gene in 15 clinical lung cancers (lung ADC, lung SCC, and SCLC; top panels) and 15 lung-cancer cell lines (bottom panels), detected by semiquantitative RT-PCR analysis. Part B depicts the expression of ADAMTS18 gene in 10 clinical ESCCs (top panels), and 10 esophageal cancer cell lines (bottom panels), detected by semiquantitative RT-PCR analysis.
Fig. 1 depicts the expression of ADAMTS18 in lung and esophageal cancers and normal tissues: Part C depicts the expression of ADAMTS18 gene in cancerous tissues and normal tissues of 5 clinical ESCCs, detected by semiquantitative RT-PCR analysis. Part D depicts the expression of ADAMTS18 in normal human tissues, detected by Northern-blot analysis.
Fig. 2 depicts the inhibition of growth of NSCLC and ESCC cells by siRNAs against ADAMTS18: The top panels depict the results of semiquantitative RT-PCR, confirming the expression of ADAMTS18 in response to siRNAs for ADAMTS18 (si-ADAMTS18-#1 or #2) or control siRNAs (EGFP or LUC) in DMS114 and TE4 cells, analyzed by semiquantitative RT-PCR. The middle and bottom panels depict the results of MTT and colony formation assays of the tumor cells transfected with si-ADAMTS18s or control siRNAs.
Fig. 3 depicts the effects of exogenous ADAMTS18 on cell growth: In Part A, transient expression of exogenous ADAMTS18 in COS-7 cells detected by Western-blotting is depicted in the left panels. The results of MTT assays of COS-7 cells transfected with ADAMTS18 cDNA or control siRNA are depicted in the right panel.
Fig. 3 depicts the effects of exogenous ADAMTS18 on cell growth: In Part B, the effect of exogenous ADAMTS18 on cell growth is depicted. Transient expression of exogenous ADAMTS18 in A549 cells detected by Western-blotting is depicted in the left panels and the results of MTT assays of A549 cells transfected with ADAMTS18 cDNA or control siRNA are depicted in the right panel.
Fig. 4 depicts the post-translational modification, subcellular localization and secretion into culture medium of exogenous ADAMTS18 protein: In Part A, the results of western-blotting of exogenous ADAMTS18 protein after incubation with N-glycosidase are depicted. In Part B, the subcellular localization of exogenous ADAMTS18 protein in COS-7 cells detected by ANTI-FLAG co-immunostained with DAPI is depicted.
Fig. 4 depicts the post-translational modification, subcellular localization and secretion into culture medium of exogenous ADAMTS18 protein: In Part C, the expression of ADAMTS18 in COS-7 cells detected by semiquantitative RT-PCR analysis is depicted. In Part D, the secretion of exogenous ADAMTS18 protein into culture medium, detected by Western-blot analysis is depicted.
Fig. 5 depicts the enhancement of cellular invasiveness induced by the introduction of ADAMTS18 into mammalian cells and MMP activity of exogenous or endogenous ADAMTS18: In Part A, the result of assays demonstrating the invasive nature of COS-7 cells in Madrigal matrix after transfection of ADAMTS18-expressing plasmids are depicted. Giemsa staining (magnification, x100; lower panels) and the relative number of cells invading through the Matrigel-coated filters (upper panel) are shown. Assays were done twice and in duplicate wells.
Fig. 5 depicts the enhancement of cellular invasiveness induced by the introduction of ADAMTS18 into mammalian cells and MMP activity of exogenous or endogenous ADAMTS18: In Part B, the results of cell growth assays, performed at the same time with Matrigel assays, are depicted. In Part C, the MMP activity of exogenous ADAMTS18 in COS-7 cells, evaluated by an MMP assay kit is depicted. In Part D, the suppression of the MMP activity of endogenous ADAMTS18 in DMS114 cells by si-ADAMTS18#1 is depicted.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
The disclosure of each publication, patent or patent application mentioned in this specification is specifically incorporated by reference herein in its entirety. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
Definitions
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated. The terms "isolated" and "purified" used in relation with a substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicates that the substance is substantially free from at least one substance that may else be included in the natural source. Thus, an isolated or purified antibody refers to antibodies that are substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In a preferred embodiment, antibodies and polypeptides of the present invention are isolated or purified. An "isolated" or "purified" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a preferred embodiment, nucleic acid molecules encoding antibodies of the present invention are isolated or purified.
The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids.
Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
The terms "gene", "polynucleotides", "oligonucleotide", "nucleotides", "nucleic acids", and "nucleic acid molecules" are used interchangeably unless otherwise specifically indicated and are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers. The polynucleotide, oligonucleotide, nucleotides, nucleic acids, or nucleic acid molecules may be composed of DNA, RNA or a combination thereof.
Unless otherwise defined, the term "cancer" refers to cancer over-expressing the ADAMTS18 gene, in particular, lung cancer, including adenocarcinoma (ADC), squamous-cell carcinoma (SCC), large-cell carcinoma (LCC), and small-cell lung cancer (SCLC) and, esophageal cancer.
As used herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene, including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)). Herein, "double-stranded molecule" is also referred to as "double-stranded nucleic acid ", " double-stranded nucleic acid molecule", "double-stranded polynucleotide", "double-stranded polynucleotide molecule", "double-stranded oligonucleotide" and "double-stranded oligonucleotide molecule".
As used herein, the term "target sequence" refers to a nucleotide sequence within mRNA or cDNA sequence of a target gene, which will result in suppression of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene. A nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded molecule including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. When a target sequence is shown by cDNA sequence, a sense strand sequence of a double-stranded cDNA, i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence. A double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule. Herein, the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule. For example, when a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base "t"s within the RNA region is replaced with base "u"s. On the other hand, when a target sequence is shown in an RNA sequence and a sense strand of a double-stranded molecule has a DNA region, base "u"s within the DNA region is replaced with "t"s. For example, when a target sequence is shown in the RNA sequence of SEQ ID NO: 11 or 12 and the sense strand of the double-stranded molecule has the 3' side half region composed of DNA, "a sequence corresponding to a target sequence" is "5'-GCCAGUAUCUCAAGAAATT-3'" (for SEQ ID NO: 11) or "5'-GGGCACAACUTTGGUATGA-3'" (for SEQ ID NO: 12). Also, a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand. For example, when a target sequence is shown in the RNA sequence of SEQ ID NO: 11 or 12 and the antisense strand of the double-stranded molecule has the 5' side half region composed of DNA, "a complementary sequence to a target sequence" is 3'- CGGUCAUAGAGTTCTTTAA -5'" (for SEQ ID NO: 11) or "3'- CCCGUGUUGAAACCATACT -5'" (for SEQ ID NO: 12).
On the other hand, when a double-stranded molecule is composed of RNA, the sequence corresponding to a target sequence shown in SEQ ID NO: 11 or 12 is the RNA sequence of SEQ ID NO: 11 or 12, and the complementary sequence corresponding to a target sequence shown in SEQ ID NO: 11 or 12 is the RNA sequence of "3'- CGGUCAUAGAGUUCUUUAA-5'" (for SEQ ID NO:11) or "3'- CCCGUGUUGAAACCAUACU-5'" (for SEQ ID NO:12).
A double-stranded molecule may have one or two 3' overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
As used herein, the term "siRNA" refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a sense nucleic acid sequence (also referred to as "sense strand"), an antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin. The siRNA may either be a dsRNA or shRNA.
As used herein, the term "dsRNA" refers to a construct of two RNA molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
The term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
As used herein, the term "siD/R-NA" refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes a sense nucleic acid sequence (also referred to as "sense strand"), an antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin. The siD/R-NA may either be a dsD/R-NA or shD/R-NA.
As used herein, the term "dsD/R-NA" refers to a construct of two molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene. One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
The term "shD/R-NA", as used herein, refers to an siD/R-NA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
As used herein, an "isolated nucleic acid" is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. In the context of the present invention, examples of isolated nucleic acid include DNA, RNA, and derivatives thereof.
I. ADAMTS18 polynucleotide and ADAMTS18 polypeptide
The present invention is based in part on the discovery of elevated expression of the ADAMTS18 gene in lung and esophageal cancer cells obtained from diseased patients. The nucleotide sequence of the human ADAMTS18 gene is shown in SEQ ID NO: 1 and is also available as GenBank Accession No. NM_199355. Herein, the ADAMTS18 gene encompasses the human ADAMTS18 gene as well as those of other animals including, but not limited to, non-human primate, mouse, rat, dog, cat, horse, and cow, and further includes allelic mutants and genes found in other animals as corresponding to the ADAMTS18 gene.
The amino acid sequence encoded the human ADAMTS18 gene is shown in SEQ ID NO: 2 and is also available as GenBank Accession No. NP_955387.1. In the context of the present invention, the polypeptide encoded by the ADAMTS18 gene is referred to as "ADAMTS18", and sometimes as "ADAMTS18 polypeptide" or "ADAMTS18 protein".
According to an aspect of the present invention, functional equivalents are also included in the ADAMTS18 protein. Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptides that retain the biological ability of the ADAMTS18 protein may be used as such functional equivalents of each protein in the present invention.
The biological activities of the ADAMTS18 protein include, for example, regulating activity for cell differentiation and cancer cell proliferation activity (cancer cell proliferation enhancing activity). Further, the biological activity of the ADAMTS18 protein may include N-glycosylation activity, invasive activity and MMP (Matrix metalloproteinase) activity.
Such functional equivalents include those that one or more amino acids are substituted, deleted, added, and/or inserted to the natural occurring amino acid sequence of the ADAMTS18 protein. Alternatively, the polypeptide may be one that includes an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the ADAMTA18 protein (e.g., SEQ ID NO: 2),, more preferably at least about 90% to 95% homology, even more preferably 96%, 97%, 98% or 99% homology. In other embodiments, the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the naturally occurring nucleotide sequence of the ADAMTS18 gene.
The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times of background, preferably 10 times of background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.
In the context of the present invention, a condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the human ADAMTS18 protein can be routinely selected by a person skilled in the art. For example, hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer. The following washing step can be conducted, for example, in a low stringent condition. An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, preferably 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions are often preferably used. An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min. However, several factors, such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
In general, modifications of one, two, or more amino acids in a protein will not influence the function of the protein. In fact, mutated or modified proteins (i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition) have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)). Accordingly, one of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence that alter a single amino acid or a small percentage of amino acids or those considered to be a "conservative modification" wherein the alteration of a protein results in a protein with similar functions, are acceptable in the context of the instant invention. Thus, in one embodiment, the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in the human ADAMTS18 sequence.
So long as the activity of the protein is maintained, the number of amino acid mutations is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 5 or 6 amino acids or less, and even more preferably 3 or 4 amino acids or less.
An amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Aspargine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
Such conservatively modified polypeptides are included in the present ADAMTS18 protein. However, the present invention is not restricted thereto and the ADAMTS18 protein includes non-conservative modifications so long as they retain at least one biological activity of the ADAMTS18 protein. Furthermore, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
Moreover, the ADAMTS18 gene of the present invention encompasses polynucleotides that encode such functional equivalents of the ADAMTS18 protein.
II. Diagnosing cancer
II-1. Method for diagnosing cancer or a predisposition for developing cancer
The expression of the ADAMTS18 gene was found to be specifically elevated in patients with cancer, more particularly, in lung and esophageal cancer cells. Accordingly, the ADAMTS18 gene, as well as its transcription and translation products, find diagnostic utility as a marker for a cancer overexpressing ADAMTS18 gene, such as lung and esophageal cancer. Moreover, by measuring the expression of the ADAMTS18 gene in as subject-derived biological sample, such as a cell sample, lung cancer or esophageal cancer can be diagnosed or detected. Such diagnosis or detection may be performed by comparing the expression level of ADAMTS18 gene between a subject-derived sample and a normal sample. More particularly, the present invention provides a method for detecting or diagnosing cancer and/or a predisposition for developing cancer in a subject by determining the expression level of the ADAMTS18 gene in the subject-derived biological sample. Preferred cancers to be diagnosed or detected by the present method include lung and esophageal cancer. In the context of the present invention, "lung cancer" includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Likewise, "NSCLC" includes adenocarcinoma, squamous cell carcinoma (SCC) and large-cell carcinoma.
Alternatively, the present invention provides a method for detecting or identifying cancer cells in a subject-derived lung or esophageal tissue sample, the method including the step of determining the expression level of the ADAMTS18 gene in a subject-derived tissue sample, wherein an increase in said expression level as compared to a normal control level indicates the presence or suspicion of cancer cells in the tissue.
Such result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease or is predisposed to developing the disease. Alternatively, the present invention may provide a doctor with useful information to diagnose that the subject suffers from the disease. For example, according to the present invention, when the suspicion or doubt of the presence of cancer cells in the tissue obtained from a subject is indicated, clinical decisions would be made by a doctor with consideration of this observation and another aspect including the pathological finding of the tissue, levels of known tumor marker(s) in blood, or clinical course of the subject, etc. For example, some well-known diagnostic lung cancer markers in blood include ACT, BFP, CA19-9, CA50, CA72-4, CA130, CA602, CEA, IAP, KMO-1, SCC, SLX, SP1, Span-1, STN, TPA, and cytokeratin 19 fragment. Alternatively, diagnostic esophageal tumor markers in blood such as CEA, DUPAN-2, IAP, NSE, SCC, SLX and Span-1 are also well known. Namely, in a particular embodiment, according to the present invention, an intermediate result for examining the condition of a subject may also be provided.
In another embodiment, the present invention provides a method for detecting a diagnostic marker of cancer, the method including the step of detecting the expression of the ADAMTS18 gene in a subject-derived biological sample as a diagnostic marker of cancer. Preferable cancers to be diagnosed by the present method include lung cancer and esophageal cancer.
In the context of the present invention, the term "diagnosing" is intended to encompass predictions and likelihood analysis. The present method is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance for cancer. According to the present invention, an intermediate result for examining the condition of a subject may also be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to determine that a subject suffers from the disease. Alternatively, the present invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
A subject to be diagnosed by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, human, non-human primate, mouse, rat, dog, cat, horse, and cow.
It is preferred to collect a biological sample from the subject to be diagnosed to perform the diagnosis. Any biological material can be used as the biological sample for the determination so long as it can include the objective transcription or translation product of the ADAMTS18 gene due to cancer. The biological samples include, but are not limited to, bodily tissues and fluids, such as blood, sputum, and urine. Preferably, the biological sample contains a cell population including an epithelial cell, more preferably a lung or esophageal epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cells may be purified from the obtained bodily tissues and fluids, and then used as the biological sample. Alternatively, biological sample may be a tissue sample collected from an area suspected to be cancerous. Preferably, the tissue sample may be a lung tissue sample or esophageal tissue sample.
According to the present invention, the expression level of the ADAMTS18 gene is determined in a subject-derived biological sample. The expression level can be determined at the transcription product (i.e., mRNA) level, using methods known in the art. For example, the mRNA of the ADAMTS18 gene may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a filter, a chip or an array. The use of an array is preferable for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including the ADAMTS18 gene. Those skilled in the art can prepare such probes utilizing the sequence information of the ADAMTS18 gene. For example, the cDNA of the ADAMTS18 gene may be used as the probes. If necessary, the probe may be labeled with a suitable label, such as dyes and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
Furthermore, the transcription product of the ADAMTS18 gene may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers used in the Example (SEQ ID NOs: 3 and 4) may be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the ADAMTS18 gene. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
The probes or primers may be of specific sizes. The sizes are selected from the group consisting of at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides and the probes and primers may range in size from 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides and 25-30 nucleotides.
Alternatively, the translation product (i.e., protein) of the ADAMTS18 gene may be detected for the diagnosis or detection of the present invention. For example, the quantity of the ADAMTS18 protein may be determined. A method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the protein. The antibody may be monoclonal or polyclonal. Furthermore, any antibody fragments or modified antibodies (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) may be used for the detection, so long as they retain the binding ability to the ADAMTS18 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
As another method to detect the expression level of the ADAMTS18 gene based on its translation product, the intensity of staining may be observed via immunohistochemical analysis using an antibody against the ADAMTS18 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of the ADAMTS18 gene.
Furthermore, the translation product may be detected based on its biological activity. Specifically, the ADAMTS18 protein was demonstrated herein to be involved in the growth of cancer cells. Thus, the cancer cell growth promoting ability of the ADAMTS18 protein may be used as an index of the ADAMTS18 protein existing in the biological sample. Herein, cell growth promoting ability is also referred to as "cell proliferative activity" or "cell proliferation enhancing activity".
Moreover, in addition to the expression level of the ADAMTS18 gene, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in lung or esophageal cancer, may also be determined to improve the accuracy of the diagnosis.
As noted above, the methods of the present invention compare the expression level of the ADAMTS18 gene in a subject-derived biological sample with that of a "control level".
In the context of the present invention, the phrase "control level" refers to the expression level of the ADAMTS18 gene detected in a control sample and encompasses both a normal control level and a cancer control level. The phrase "normal control level" refers to a level of the ADAMTS18 gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer. A normal individual is one with no clinical symptom of lung and esophageal cancer. A normal control level can be determined using a normal cell obtained from a non-cancerous tissue. A "normal control level" may also be the expression level of the ADAMTS18 gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from lung cancer or esophageal cancer. On the other hand, the phrase "cancer control level" refers to an expression level of the ADAMTS18 gene detected in the cancerous tissue or cell of an individual or population suffering from lung or esophageal cancer.
An increase in the expression level of the ADAMTS18 gene detected in a subject-derived sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing lung cancer or esophageal cancer. In the context of the present invention, the subject-derived sample may be any tissues obtained from test subjects, e.g., patients known to have or suspected of having cancer. For example, tissues may include epithelial cells. More particularly, tissues may be cancerous epithelial cells.
Alternatively, the expression level of the ADAMTS18 gene in a sample can be compared to a cancer control level of the ADAMTS18 gene. A similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer.
Herein, gene expression levels are deemed to be "increased" when the gene expression increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level. Accordingly, the expression level of cancer marker genes including the ADAMTS18 gene in a biological sample can be considered to be increased if it increases from the normal control level of the corresponding cancer marker gene by, for example, 10% or more, 25% or more, or 50% or more; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more. The expression level of the target gene can be determined by detecting, e.g., determined by the hybridization intensity of nucleic acid probes to gene transcripts in a sample.
The control level may be determined at the same time with a test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the ADAMTS18 gene in samples from subjects whose disease state are known. Furthermore, the control level can be a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of the ADAMTS18 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample. Moreover, it is preferred, to use the standard value of the expression levels of the ADAMTS18 gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.
In sum, a control level determined from a biological sample that is known to be non-cancerous is called "normal control level". On the other hand, if the control level is determined from a cancerous biological sample, it will be called "cancerous control level". In the context of the present invention, when the expression level of the ADAMTS18 gene is increased as compared to a normal control level or is similar to a cancerous control level, the subject may be diagnosed to be suffering from or at a risk of developing cancer. Furthermore, in case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
Difference between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes. Genes whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
Furthermore, the present invention provides the use of the ADAMTS18 gene as cancerous markers. The ADAMTS18 gene are particularly useful for lung and esophageal cancerous markers. For example, it can be determined whether a biological sample contains cancerous cells, especially lung or esophageal cancerous cells, by detecting the expression level of the ADAMTS18 gene as cancerous markers. Specifically, increasing the expression level of the ADAMTS18 gene in a biological sample as compared to a normal control level indicates that the biological sample contains cancerous cells. The expression level of the ADAMTS18 gene can be determined by detecting the transcription or translation products of the gene as described above.
II-2. Assessing efficacy of cancer treatment
The ADAMTS18 gene differentially expressed between normal and cancerous cells also allow for the course of cancer treatment to be monitored, and the above-described method for diagnosing cancer can be applied for assessing the efficacy of a treatment on cancer. Specifically, the efficacy of a treatment on cancer can be assessed by determining the expression level of the ADAMTS18 gene in a cell(s) derived from a subject undergoing the treatment. If desired, test cell populations are obtained from the subject at various time points, before, during, and/or after the treatment. The expression level of the ADAMTS18 gene can be, for example, determined following the method described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer. In the context of the present invention, it is preferable that the control level to which the detected expression level is compared be obtained from the ADAMTS18 gene expression in a cell(s) not exposed to the treatment of interest.
If the expression level of the ADAMTS18 gene is compared to a control level that is obtained from a normal cell or a cell population containing no cancer cell, a similarity in the expression level indicates that the treatment of interest is efficacious and a difference in the expression level indicates less favorable clinical outcome or prognosis of that treatment. On the other hand, if the comparison is conducted against a control level that is obtained from a cancer cell or a cell population containing a cancer cell(s), a difference in the expression level indicates efficacious treatment, while a similarity in the expression level indicates less favorable clinical outcome or prognosis.
Furthermore, the expression levels of the ADAMTS18 gene before and after a treatment can be compared according to the present method to assess the efficacy of the treatment. Specifically, the expression level detected in a subject-derived biological sample after a treatment (i.e., post-treatment level) is compared to the expression level detected in a biological sample obtained prior to treatment onset from the same subject (i.e., pre-treatment level). A decrease in the post-treatment level compared to the pre-treatment level indicates that the treatment of interest is efficacious while an increase in or similarity of the post-treatment level to the pre-treatment level indicates less favorable clinical outcome or prognosis.
As used herein, the term "efficacious" indicates that the treatment leads to a reduction in the expression of a pathologically up-regulated gene, an increase in the expression of a pathologically down-regulated gene or a decrease in size, prevalence, or metastatic potential of carcinoma in a subject. When a treatment of interest is applied prophylactically, "efficacious" means that the treatment retards or prevents the forming of tumor or retards, prevents, or alleviates at least one clinical symptom of cancer. Assessment of the state of tumor in a subject can be made using standard clinical protocols.
In addition, efficaciousness of a treatment can be determined in association with any known method for diagnosing cancer. Cancers can be diagnosed, for example, by identifying symptomatic anomalies, e.g., weight loss, abdominal pain, back pain, anorexia, nausea, vomiting and generalized malaise, weakness, and jaundice.
To the extent that the methods and compositions of the present invention find utility in the context of "prevention" and "prophylaxis", such terms are interchangeably used herein to refer to any activity that reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels." While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g., reducing the proliferation and metastasis of tumors.
The treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis. Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer. For example, reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis includes 10%, 20%, 30% or more reduction, or stable disease.
III. Kits and reagents
The present invention also provides reagents for detecting or diagnosing cancer, i.e., reagents that can detect the transcription or translation product of the ADAMTS18 gene. Examples of such reagents include those capable of:
(a) detecting mRNA of the ADAMTS18 gene;
(b) detecting the ADAMTS18 protein; and/or
(c) detecting the biological activity of the ADAMTS18 protein, in a subject-derived biological sample.
Suitable reagents include nucleic acids that specifically bind to or identify a transcription product of the ADAMTS18 gene. For example, a nucleic acid that specifically binds to or identifies a transcription product of the ADAMTS18 gene includes, for example, oligonucleotides (e.g., probes and primers) having a sequence that is complementary to a portion of the ADAMTS18 gene transcription product. Such oligonucleotides are exemplified by primers and probes that are specific to the mRNA of the gene of interest and may be prepared based on methods well known in the art. Alternatively, antibodies can be exemplified as reagents for detecting the translation product of the gene. The probes, primers, and antibodies described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer' can be mentioned as suitable examples of such reagents. These reagents may be used for the above-described diagnosis or detection of cancer, particularly lung cancer and esophageal cancer. The assay format for using the reagents may be Northern hybridization or sandwich ELISA, both of which are well-known in the art.
The detection reagents may be packaged together in the form of a kit. For example, the detection reagents may be packaged in separate containers. Furthermore, the detection reagents may be packaged with other reagents necessary for the detection. For example, a kit may include a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix) as the detection reagent, a control reagent (positive and/or negative), and/or a detectable label. A kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes. These reagents and such may be retained in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay may also be included in the kit.
Although the present kit is suited for the detection and diagnosis of lung and esophageal cancer, it may also be useful in assessing the prognosis of cancer and/or monitoring the efficacy of a cancer therapy.
As an aspect of the present invention, the reagents for diagnosing or detecting cancer may be immobilized on a solid matrix, such as a porous strip, to form at least one site for detecting cancer. The measurement or detection region of the porous strip may include a plurality of sites, each containing a detection reagent (e.g., nucleic acid). A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a separate strip from the test strip. Optionally, the different detection sites may contain different amounts of immobilized detection reagents (e.g., nucleic acid), i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test biological sample, the number of sites displaying a detectable signal provides a quantitative indication of the expression level of the ADAMTS18 gene in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
IV. Screening methods
Using the ADAMTS18 gene, a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene, it is possible to screen substances that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene. Such substances may be used as pharmaceuticals for treating or preventing cancer, in particular, lung and esophageal cancer. Thus, the present invention provides methods of screening for candidate substances for treating or preventing cancer using the ADAMTS18 gene, a polypeptide encoded by the gene or fragments thereof, or a transcriptional regulatory region of the gene.
A substance isolated by the screening method of the present invention is a substance that is expected to inhibit the expression of the ADAMTS18 gene, or the activity of the translation product of the gene, and thus, is a candidate for treating or preventing diseases attributed to, for example, cell proliferative diseases, such as cancer (in particular, lung and esophageal cancer). Namely, the substances screened through the present methods are deemed to have a clinical benefit and can be further tested for its ability to prevent cancer cell growth in animal models or test subjects.
In the context of the present invention, substances to be identified through the present screening methods may be any compound or composition including several compounds. Furthermore, a test substance exposed to a cell or protein in the screening methods of the present invention may be a single compound or a combination of compounds. When a combination of compounds is used in the methods, the compounds may be contacted sequentially or simultaneously.
Any test substances, for example, cell extracts, cell culture supernatants, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention. Test substances useful in the screenings described herein can also be antibodies that specifically bind to a protein of interest or a partial peptide thereof that lacks the biological activity of the original proteins in vivo.
Test substances of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including:
(1) biological libraries,
(2) spatially addressable parallel solid phase or solution phase libraries,
(3) synthetic library methods requiring deconvolution,
(4) the "one-bead one-compound" library method and
(5) synthetic library methods using affinity chromatography selection.
The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of compounds may be presented in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Pat. Application 2002103360).
Although the construction of test substance libraries is well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.
It is herein revealed that suppression of the expression level and/or biological activity of ADAMTS18 lead to suppression of the growth of cancer cells. Therefore, when a substance suppresses the expression and/or activity of ADAMTS18, such suppression is indicative of a potential therapeutic effect in a subject. In the context of the present invention, a potential therapeutic effect refers to a clinical benefit with a reasonable expectation. Examples of such clinical benefits include but are not limited to:
(a) reduction in expression of the ADAMTS18 gene,
(b) a decrease in size, prevalence, or metastatic potential of the cancer in the subject,
(c) preventing cancers from forming, or
(d) preventing or alleviating a clinical symptom of cancer.
A. Molecular Modeling:
Construction of test substance libraries is facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of ADAMTS18 protein. One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.
Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
An example of the molecular modeling system described generally above includes the CHARMm and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
A number of articles have been published on the subject of computer modeling of drugs interactive with specific proteins, examples of which include Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and, with respect to a model receptor for nucleic acid components, Askew et al., J Am Chem Soc 1989, 111: 1082-90.
Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See, e.g., DesJarlais et al., J Med Chem 1988, 31: 722-9; Meng et al., J Computer Chem 1992, 13: 505-24; Meng et al., Proteins 1993, 17: 266-78; Shoichet et al., Science 1993, 259: 1445-50.
Once a putative inhibitor has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below. The resulting library of putative inhibitors may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, particularly lung and esophageal cancer.
B. Combinatorial Chemical Synthesis:
Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively, simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library. An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
Preparation of combinatorial chemical libraries is well known to those of skill in the art, and may be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates (Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries (see Ausubel, Current Protocols in Molecular Biology 1995 supplement; Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22; US Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1;6(6):624-31.; isoprenoids, US Patent 5,569,588; thiazolidinones and metathiazanones, US Patent 5,549,974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morpholino compounds, US Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).
C. Other Candidates:
Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 106 -108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples. Furka et al. (14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93), Houghten (US Patent 4,631,211) and Rutter et al. (US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target. Tuerk and Gold (Science. 249:505-510 (1990)) disclose SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers. In the SELEX method, a large library of nucleic acid molecules (e.g., 1015 different molecules) can be used for screening.
A compound in which a part of the structure of the compound screened by any of the present screening methods is converted by addition, deletion and/or replacement, is included in the substances obtained by the screening methods of the present invention.
Furthermore, when the screened test substance is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. The obtained DNA finds use in preparing the test substance which is a candidate for treating or preventing cancer.
IV-1. Protein based screening methods
According to the present invention, the expression of the ADAMTS18 gene is crucial for the growth and/or survival of cancer cells, in particular lung and esophageal cancer cells. Furthermore, the ADAMTS18 protein has been demonstrated to have N-glycosylation activity, invasive activity as well as MMP (Matrix metalloproteinase) activity in the present invention. Accordingly, substances that suppress the function of the ADAMTS18 polypeptide would be presumed to inhibit the growth and/or survival of cancer cells, and therefore find use in treating or preventing cancer. Thus, the present invention provides methods of screening a candidate substance for treating or preventing cancer, using the ADAMTS18 polypeptide. Further, the present invention also provides methods of screening a candidate substance for inhibiting the growth and/or survival of cancer cells, using the ADAMTS18 polypeptide.
In addition to the ADAMTS18 polypeptide, fragments of the polypeptides may be used for the present screening, so long as it retains at least one biological activity of the naturally occurring ADAMTS18 polypeptide. In the preferred embodiment, a fragment of the ADAMTS18 polypeptide may include a metalloproteinase domain of the polypeptide (e.g., 293 to 494 of SEQ ID NO: 2). Alternatively, mature polypeptide of the ADAMTS18 (e.g., 285 to 1221 of SEQ ID NO: 2) may be used for the screening method of the present invention.
The ADAMTS18 protein may be produced in vitro by means of an in vitro translation system.
The ADAMTS18 polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.
The polypeptides or fragments used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
6) WO99/67288; and
7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
Alternatively, the proteins may be obtained through any known genetic engineering methods for producing polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, a suitable vector including a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence including a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the ADAMTS18 polypeptide is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8. A promoter may be used for the expression. Any commonly used promoters may be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SRalpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such. The introduction of the vector into host cells to express the ADAMTS18 gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.
The polypeptides or fragments thereof may be further linked to other substances, so long as the polypeptides and fragments retain at least one biological activity. Usable substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. Such modifications may be used to confer additional functions or to stabilize the polypeptide and fragments.
For example, the polypeptides may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide. Alternatively, a commercially available epitope-antibody system may be used (Experimental Medicine 13: 85-90 (1995)). Vectors that are capable of expressing a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and so on, by the use of its multiple cloning sites are commercially available.
A fusion protein, prepared by introducing only small epitopes composed of several to a dozen amino acids so as not to change the property of the original polypeptide by the fusion, is also provided herein. Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and antibodies recognizing them may be used as the epitope-antibody system for detecting the binding activity between the polypeptides (Experimental Medicine 13: 85-90 (1995)).
IV-1-1. Identifying substances that bind to the polypeptides
A substance that binds to a protein is likely to alter the expression of the gene coding for the protein or the biological activity of the protein. Thus, as an aspect, the present invention provides a method of screening a candidate substance for treating or preventing cancer, which includes steps of:
a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof;
b) detecting binding (or binding activity) between the polypeptide or fragment and the test substance; and
c) selecting the test substance that binds to the polypeptide as a candidate substance for treating or preventing cancer.
According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing ADAMTS18 associated disease (e.g., cancer) may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing ADAMTS18 associated disease (e.g., cancer), using the ADAMTS18 polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof;
b) detecting the binding (or binding activity) between the polypeptide or fragment and the test substance; and
c) correlating the binding of b) with the therapeutic effect of the test substance or compound.
Alternatively, according to the present invention, the potential therapeutic effect of a test substance or compound on treating or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide of ADAMTS18;
(b) detecting the binding activity between the polypeptide and the test substance; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance binds to the polypeptide.
In the context of the present invention, the therapeutic effect may be correlated with the binding level to ADAMTS18 polypeptide or a functional fragment thereof. For example, when the test substance binds to ADAMTS18 polypeptide or a functional fragment thereof, the test substance may identified or selected as the candidate substance having the requisite therapeutic effect. Alternatively, when the test substance does not bind to an ADAMTS18 polypeptide or a functional fragment thereof, the test substance may identified as the substance having no significant therapeutic effect.
In the present invention, it is revealed that suppressing the expression of ADAMTS18 reduces cancer cell growth. Thus, by screening for candidate substances that binds to ADAMTS18 polypeptide, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
The binding of a test substance to the ADAMTS18 polypeptide may be, for example, detected by immunoprecipitation using an antibody against the polypeptide. Therefore, for the purpose for such detection, it is preferred that the ADAMTS18 polypeptide or fragments thereof used for the screening contains an antibody recognition site. The antibody used for the screening may be one that recognizes an antigenic region (e.g., epitope) of the ADAMTS18 polypeptide. Preparation methods for such antibodies are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
Alternatively, the ADAMTS18 polypeptide or a fragment thereof may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody at its N- or C-terminus. The specificity of the antibody has been revealed, to the N- or C- terminus of the polypeptide. A commercially available epitope-antibody system can be used (Experimental Medicine 1995, 13:85-90). Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and such by the use of its multiple cloning sites are commercially available and can be used for the present invention. Furthermore, fusion proteins containing much smaller epitopes to be detected by immunoprecipitation with an antibody against the epitopes are also known in the art (Experimental Medicine 1995, 13:85-90). Such epitopes, composed of several to a dozen amino acids so as not to change the property of the ADAMTS18 polypeptide or fragments thereof, can also be used in the present invention. Examples include polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), and such and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the ADAMTS18 polypeptide (Experimental Medicine 13: 85-90 (1995)).
Glutathione S-transferase (GST) is also well-known as the counterpart of the fusion protein to be detected by immunoprecipitation. When GST is used as the protein to be fused with the ADAMTS18 polypeptide or fragment thereof to form a fusion protein, the fusion protein can be detected either with an antibody against GST or a substance specifically binding to GST, i.e., such as glutathione (e.g., glutathione-Sepharose 4B).
In immunoprecipitation, an immune complex is formed by adding an antibody (recognizing the ADAMTS18 polypeptide or a fragment thereof itself, or an epitope tagged to the polypeptide or fragment) to the reaction mixture of the ADAMTS18 polypeptide and the test substance. If the test substance has the ability to bind the polypeptide, then the formed immune complex will consist of the ADAMTS18 polypeptide, the test substance, and the antibody. On the contrary, if the test substance is devoid of such ability, then the formed immune complex only consists of the ADAMTS18 polypeptide and the antibody. Therefore, the binding ability of a test substance to ADAMTS18 polypeptide can be examined by, for example, measuring the size of the formed immune complex. Any method for detecting the size of a compound can be used, including chromatography, electrophoresis, and such. For example, when mouse IgG antibody is used for the detection, Protein A or Protein G sepharose can be used for quantitating the formed immune complex.
For more details on immunoprecipitation see, for example, Harlow et al., Antibodies, Cold Spring Harbor Laboratory publications, New York, 1988, 511-52. SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Detection may be achieved using conventional staining method, such as Coomassie staining or silver staining, or, for proteins that is difficult to detect, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35S-methionine or 35S-cysteine, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
Furthermore, the ADAMTS18 polypeptide or a fragment thereof used for the screening of substances that bind thereto may be bound to a carrier. Example of carriers that may be used for binding the polypeptides include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercially available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column. Alternatively, the use of magnetic beads is also known in the art, and enables to readily isolate polypeptides and substances bound on the beads via magnetism.
The binding of a polypeptide to a carrier may be conducted according to routine methods, such as chemical bonding and physical adsorption. Alternatively, a polypeptide may be bound to a carrier via antibodies specifically recognizing the protein. Moreover, binding of a polypeptide to a carrier can also be conducted by means of interacting molecules, such as the combination of avidin and biotin.
Screening using such carrier-bound ADAMTS18 polypeptide or fragments thereof include, for example, contacting a test substance to the carrier-bound polypeptide, incubating the mixture, washing the carrier, and detecting and/or measuring the substance bound to the carrier. The binding may be carried out in buffer, for example, but are not limited to, phosphate buffer and Tris buffer, as long as the buffer does not inhibit the binding.
When such a carrier-bound ADAMTS18 polypeptide or fragments thereof, and a composition (e.g., cell extracts, cell lysates, etc.) are used as the test substance in a screening method, such method is generally called affinity chromatography. For example, the ADAMTS18 polypeptide may be immobilized on a carrier of an affinity column, and a test substance, containing a substance capable of binding to the polypeptides, is applied to the column. After loading the test substance, the column is washed, and then the substance bound to the polypeptide is eluted with an appropriate buffer.
A biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention. When such a biosensor is used, the interaction between the ADAMTS18 polypeptide and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of the polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide and test substance using a biosensor such as BIAcore.
Methods of screening for molecules that bind to a specific protein among synthetic chemical compounds, or molecules in natural substance banks or a random phage peptide display library by exposing the specific protein immobilized on a carrier to the molecules, and methods of high-throughput screening based on combinatorial chemistry techniques (Wrighton et al., Science 1996, 273:458-64; Verdine, Nature 1996, 384:11-3) to isolate not only proteins but chemical compounds are also well-known to those skilled in the art. These methods can also be used for screening substances (including agonist and antagonist) that bind to the ADAMTS18 protein or fragments thereof.
When the test substance is a protein, for example, West-Western blotting analysis (Skolnik et al., Cell 1991, 65:83-90) can be used for the present method. Specifically, a protein binding to the ADAMTS18 polypeptide can be obtained by preparing first a cDNA library from cells, tissues, organs, or cultured cells (e.g., PC cell lines) expected to express at least one protein binding to the ADAMTS18 polypeptide using a phage vector (e.g., ZAP), expressing the proteins encoded by the vectors of the cDNA library on LB-agarose, fixing the expressed proteins on a filter, reacting the purified and labeled ADAMTS18 polypeptide with the above filter, and detecting the plaques expressing proteins to which the ADAMTS18 polypeptide has bound according to the label of the ADAMTS18 polypeptide.
Labeling substances such as radioisotope (e.g., 3H, 14C, 32P, 33P, 35S, 125I, 131I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucosidase), fluorescent substances (e.g., fluorescein isothiocyanate (FITC), rhodamine) and biotin/avidin, may be used for the labeling of ADAMTS18 polypeptide in the present method. When the protein is labeled with radioisotope, the detection or measurement can be carried out by liquid scintillation. Alternatively, when the protein is labeled with an enzyme, it can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.
Moreover, the ADAMTS18 polypeptide bound to the protein can be detected or measured by utilizing an antibody that specifically binds to the ADAMTS18 polypeptide or a peptide or polypeptide (for example, GST) that is fused to the ADAMTS18 polypeptide. In case of using an antibody in the present screening, the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance. Alternatively, the antibody against the ADAMTS18 polypeptide may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance. Furthermore, the antibody bound to the ADAMTS18 polypeptide in the present screening may be detected or measured using protein G or protein A column.
Antibodies to be used in the present screening methods can be prepared using techniques well known in the art. Antigens to prepared antibodies may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, rabbit, or rat, more preferably from a human. The polypeptide used as the antigen can be recombinantly produced or isolated from natural sources. The polypeptides to be used as an immunization antigen may be a complete protein or a partial peptide derived from the complete protein.
Any mammalian animal may be immunized with the antigen; however, the compatibility with parental cells used for cell fusion is preferably taken into account. In general, animals of the order Rodentia, Lagomorpha or Primate are used. Animals of the Rodentia order include, for example, mice, rats and hamsters. Animals of Lagomorpha order include, for example, hares, pikas, and rabbits. Animals of Primate order include, for example, monkeys of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkeys, sacred baboons and chimpanzees.
Methods for immunizing animals with antigens are well known in the art. Intraperitoneal injection or subcutaneous injection of antigens is a standard method for immunizing mammals. More specifically, antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc. If desired, the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals. Preferably, it is followed by several administrations of the antigen mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days. An appropriate carrier may also be used for immunization. After immunization as above, the serum is examined by a standard method for an increase in the amount of desired antibodies.
Polyclonal antibodies may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method. Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies isolated from the serum. Immunoglobulin G or M can be prepared from a fraction which recognizes only the objective polypeptide using, for example, an affinity column coupled with the polypeptide, and further purifying this fraction using protein A or protein G column.
To prepare monoclonal antibodies, immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from spleen. Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
The above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al., (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).
Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium). The cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma, to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
In addition to the above method, in which a non-human animal is immunized with an antigen for preparing hybridoma, human lymphocytes, such as those infected by the EB virus, may be immunized with an antigen, cells expressing such antigen, or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the antigen (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).
The obtained hybridomas may be subsequently transplanted into the abdominal cavity of a mouse and the ascites may be extracted. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column carrying an objective antigen.
Antibodies against the ADAMTS18 polypeptide can be used not only in the present screening method, but also for the detection of the polypeptides as cancer markers in biological samples as described in "II. Diagnosing cancer". They may further serve as candidates for agonists and antagonists of the polypeptides of interest. In addition, such antibodies, serving as candidates for antagonists, can be applied to the antibody treatment for diseases related to the ADAMTS18 polypeptide including lung and esophageal cancer as described infra.
Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)). For example, a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody. Such recombinant antibody can also be used in the context of the present screening.
Furthermore, antibodies used in the screening and so on may be fragments of antibodies or modified antibodies, so long as they retain the original binding activity. For instance, the antibody fragment may be an Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). Modified antibodies can be obtained through chemically modification of an antibody. These modification methods are conventional in the field.
Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by appropriately selected and combined column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)); however, the present invention is not limited thereto. A protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
Exemplary chromatography, with the exception of affinity, includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
Alternatively, in another embodiment of the screening method of the present invention, two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton et al., Cell 1992, 68:597-612" and "Fields et al., Trends Genet 1994, 10:286-92"). In two-hybrid system, an ADAMTS18 polypeptide or a fragment thereof is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express at least one protein binding to the ADAMTS18 polypeptide such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the ADAMTS18 polypeptide is expressed in the yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
Examples of suitable reporter genes include, but are not limited, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
The substances isolated by this screening are considered to candidates for agonists or antagonists of the ADAMTS18 polypeptide. The term "agonist" refers to molecules that activate the function of the polypeptide by binding thereto. On the other hand, the term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. Moreover, an substance isolated by this screening as an antagonist is a candidate that inhibits the in vivo interaction of the ADAMTS18 polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).
IV-1-2. Identifying substances by detecting biological activity of the polypeptides
The present invention also provides a method for screening a candidate substance for treating or preventing cancer using the ADAMTS18 polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof;
b) detecting the biological activity of the polypeptide or fragment of the step (a); and
c) selecting the test substance that reduces the biological activity of the polypeptide as compared to the biological activity in the absence of the test substance.
According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing an ADAMTS18 associated disease may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing an ADAMTS18 associated disease, using the ADAMTS18 polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with an ADAMTS18 polypeptide or a functional fragment thereof;
b) detecting the biological activity of the polypeptide or fragment of step (a); and
c) correlating the biological activity of b) with the therapeutic effect of the test substance.
Alternatively, in some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide of ADAMTS18 gene;
(b) detecting the biological activity of the polypeptide of step (a); and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the polypeptide encoded by the polynucleotide of ADAMTS18 gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.
In the present invention, the therapeutic effect may be correlated with the biological activity of an ADAMTS18 polypeptide or a functional fragment thereof. For example, when the test substance suppresses or inhibits the biological activity of an ADAMTS18 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the biological activity of ADAMTS18 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
In the present invention, it is revealed that suppressing the expression of ADAMTS18 reduces cancer cell growth. Thus, by screening for candidate substances that suppresses the biological activity of the polypeptide, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance binding to ADAMTS18 protein inhibits described above activities of the cancer, it may be concluded that such substance has the ADAMTS18 specific therapeutic effect.
Any substance can be used for the screening so long it suppresses or reduces a biological activity of the ADAMTS18 polypeptide. In the context of the instant invention, the phrase "suppress or reduce a biological activity" encompasses at least 10% suppression of the biological activity of ADAMTS18 in comparison with in the absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at least 90% suppression. Such suppression can serve an index in the present screening method.
In the preferred embodiments, control cells which do not express ADAMTS18 polypeptide are used. Accordingly, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing an ADAMTS18 associated disease, using the ADAMTS18 polypeptide or fragments thereof including the steps as follows:
a) culturing cells which express a ADAMTS18 polypeptide or a functional fragment thereof, and control cells that do not express a ADAMTS18 polypeptide or a functional fragment thereof in the presence of the test substance;
b) detecting the biological activity of the cells which express the protein and control cells; and
c) selecting the test compound that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance. According to the present invention, the ADAMTS18 polypeptide has been demonstrated to be required for the growth or viability of lung and esophageal cancer cells. The biological activities of the ADAMTS18 polypeptide that can be used as an index for the screening include such cell growth promoting activity of the human ADAMTS18 polypeptide. Herein, cell growth promoting ability is also referred to as "cell proliferative activity" or "cell proliferation enhancing activity". Also, the ADAMTS18 polypeptide has N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity. Thus, these activity may also be used as indexes for the screening.
When the biological activity to be detected in the present method is cell proliferation enhancing activity, it can be detected, for example, by preparing cells which express the ADAMTS18 polypeptide or a fragment thereof, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by detecting wound-healing activity, conducting Matrigel invasion assay and measuring the colony forming activity.
When the cell proliferation enhancing activity is being evaluated, control cells that do not express the ADAMTS18 polypeptide are preferably used. Accordingly, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer, using the ADAMTS18 polypeptide or fragments thereof including the steps as follows:
a) culturing cells which express an ADAMTS18 polypeptide or a functional fragment thereof in the presence of the test substance;
b) detecting the biological activity of the cells which express the polypeptide; and
c) selecting the test substance that inhibits the biological activity in the cells which express the polypeptide as compared to the proliferation detected in the absence of the test substance.
When the biological activity to be detected in the present method is N-glycosylation activity, it can be detected, for example, by contacting a cell expressing ADAMTS18 polypeptide or functional equivalent thereof having N-glycosylation activity, or a purified ADAMTS18 polypeptide with a test substance, and detecting N-glycosylation level of the ADAMTS18 polypeptide or the functional equivalent under a suitable condition for N-glycosylation of the ADAMTS18 polypeptide. A test substance that modulates glycosylation level of the ADAMTS18 polypeptide or functional equivalent is thereby identified.
In the context of the present invention, N-glycosylation level of an ADAMTS18 polypeptide can be determined by methods known in the art. For example, N-glycosylation of the polypeptide may be detected by comparing the molecular weight. Molecular weight of an N-glycosylated protein is larger than that of predicted size calculated from the amino acid sequence of the polypeptide by addition of glycoside chain. Methods for estimating a molecular weight of a protein are well known.
Alternatively, radiolabeled donor for N-glycosylation may be used for detection the addition of glycoside chain to the polypeptide. Transfer of the radiolabel to the ADAMTS18 protein can be detected, for example, by SDS-PAGE electrophoresis and fluorography. Alternatively, following the reaction, the ADAMTS18 polypeptides can be separated from the glycosyl donor by filtration, and the amount of radiolabel retained on the filter quantitated by scintillation counting. Other suitable labels that can be attached to glycosyl donor, such as chromogenic and fluorescent labels, and methods of detecting transfer of these labels to the ADAMTS18 polypeptide, are known in the art. Alternatively, N-glycosylation level of ADAMTS18 polypeptide can be determined by reagents that selectively recognize glycosylated site of the polypeptide. For example, after incubation of the ADAMTS18 polypeptide and a candidate substance, under the condition capable of glycosylation of the polypeptide, the N-glycosylation level of the polypeptide can be detected by immunological method. Any immunological techniques using an antibody that recognizes a glycosylated polypeptide can be used for the detection. For example, ELISA or Immunoblotting with antibodies recognizing glycosylated polypeptide can be used for the present invention.
Instead of using antibodies, glycosylated protein can be detected using reagents that selectively bind glycoside chain with high affinity. Such reagents are known in the art. For example, lectins are well known as glycoside chain specific probe. Lectin reagent conjugated with detectable label such as alkaline-phosphatase is commercially available.
The N-glycosylation level of a polypeptide in a cell may be estimated by separation of cell lysate. For example, SDS-polyacrylamide gel can be used as the separation of the polypeptide. The polypeptide separated in the gels is transferred to nitrocellulose membranes for immunoblotting analysis.
When the biological activity to be detected in connection with the present method is invasive activity, it can be detected, for example, by preparing cells that express the ADAMTS18 polypeptide and counting invasive cells number using Matrigel invasion assay, for example, as shown in Fig. 5A. The substances that reduce the invasive cell number are selected as candidate substance for treating or preventing lung or esophageal cancer.
When the biological activity to be detected in connection with the present method is MMP activity, it can be detected, for example, by contacting a cell expressing ADAMTS18 polypeptide or functional equivalent thereof, or a purified ADAMTS18 polypeptide or functional equivalent thereof with a test substances, followed by applying MMP assay to evaluate the MMP activity. The MMP assay can be conducted by methods well-known in the art. For example, the MMP assay may be conducted by using, a commercially available assay kit that includes a substrate for MMP.
The substance isolated by the present screening method is a candidate for an antagonist of the ADAMTS18 polypeptide, and thus, is a candidate that inhibits the in vivo interaction of the polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).
IV-2. Nucleotide based screening methods
IV-2-1. Screening method using ADAMTS18 gene
As discussed in detail above, by controlling the expression level of the ADAMTS18 gene, one can control the onset and progression of cancer. Thus, substances that may be used in the treatment or prevention of cancers can be identified through screenings that use the expression levels of the ADAMTS18 gene as indices. In the context of the present invention, such screening may include, for example, the following steps:
a) contacting a test substance with a cell expressing an ADAMTS18 gene;
b) detecting the expression level of the ADAMTS18 gene;
c) comparing the expression level with the expression level detected in the absence of the test substance; and
d) selecting the test substance that reduces the expression level as a candidate substance for treating or preventing cancer.
According to the present invention, the therapeutic effect of the test substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer.
In the context of the present invention, such screening may include, for example, the following steps:
a) contacting a test substance with a cell expressing an ADAMTS18 gene;
b) detecting the expression level of the ADAMTS18 gene; and
c) correlating the expression level of b) with the therapeutic effect of the test substance.
Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance in treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of:
(a) contacting a candidate substance with a cell expressing ADAMTS18; and;
(b) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance reduces the expression level of ADAMTS18 as compared to a control.
In the context of the present invention, the therapeutic effect may be correlated with the expression level of the ADAMTS18 gene. For example, when the test substance reduces the expression level of the ADAMTS18 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level of the ADAMTS18 gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
Herein, it was revealed that suppressing the expression of ADAMTS18 gene reduces cancer cell growth. Thus, by screening for candidate substance that reduces the expression level of ADAMTS18, candidate substance that have the potential to treat or prevent cancers can be identified. Potential of these candidate substance to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
A substance that inhibits the expression of the ADAMTS18 gene can be identified by contacting a cell expressing the ADAMTS18 gene with a test substance and then determining the expression level of the ADAMTS18 gene. Naturally, the identification may also be performed using a population of cells that express the gene in place of a single cell. A decreased expression level detected in the presence of a test substance as compared to the expression level in the absence of the test substance indicates the test substance as being an inhibitor of the ADAMTS18 gene, suggesting the possibility that the test substance is useful for inhibiting cancer, thus the test substance to be used for the treatment or prevention of cancer.
The expression level of a gene can be estimated by methods well known to one skilled in the art. The expression level of the ADAMTS18 gene can be, for example, determined following the method described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer'.
The cell or the cell population used for such identification may be any cell or any population of cells so long as it expresses the ADAMTS18 gene. For example, the cell or population may be or contain a lung and esophageal epithelial cell derived from a tissue. Alternatively, the cell or population may be or contain an immortalized cell derived from a carcinoma cell, including lung and esophageal cancer cell. Cells expressing the ADAMTS18 gene include, for example, cell lines established from cancers (e.g., lung and esophageal cancer cell lines such as NCI-H1781, NCI-H1373, LC319, A549, PC-14, SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170, LU61, TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, TE10, SBC-3, SBC-5, DMS114, DMS273 etc.). Furthermore, the cell or population may be or contain a cell which has been transfected with the ADAMTS18 gene.
The present method allows screening of various test substances mentioned above and is particularly suited for screening functional nucleic acid molecules including antisense RNA, siRNA, and such.
IV-2-2. Screening method using transcriptional regulatory region of ADAMTS18 gene
According to another aspect, the present invention provides a method that includes the following steps of:
a) contacting a test substance with a cell into which a vector, including a transcriptional regulatory region of an ADAMTS18 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
b) detecting the expression or activity of said reporter gene;
c) comparing the expression level or activity with the expression level or activity detected in the absence of the substance ; and
d) selecting the substance that reduces the expression or activity of said reporter gene as a candidate substance for treating or preventing cancer.
According to the present invention, the therapeutic effect of the test substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer.
According to another aspect, the present invention provides a method which includes the following steps of:
a) contacting a test substance with a cell into which a vector, composed of a transcriptional regulatory region of an ADAMTS18 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
b) detecting the expression or activity of said reporter gene; and
c) correlating the expression level of b) with the therapeutic effect of the test substance.
Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of ADAMTS18, the method including steps of:
(a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of ADAMTS18 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression or activity of said reporter gene.
In the present invention, the therapeutic effect may be correlated with the expression or activity of said reporter gene. For example, when the test substance reduces the expression or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
Herein, it was revealed that suppressing the expression of ADAMTS18 gene reduces cell growth. Thus, by screening for test substances that reduce the expression or activity of the reporter gene, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
Suitable reporter genes and host cells are well known in the art. The reporter construct required for the screening can be prepared using the transcriptional regulatory region of the ADAMTS18 gene, which can be obtained as a nucleotide segment containing the transcriptional regulatory region from a genome library based on the nucleotide sequence information of the gene.
The transcriptional regulatory region may be, for example, the promoter sequence of the ADAMTS18 gene. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of ADAMTS18 gene. The transcriptional regulatory region of ADAMTS18 gene herein is the region from start codon to at least 500 bp upstream, preferably 1,000 bp, more preferably 5,000 or 10,000 bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
When a cell(s) transfected with a reporter gene that is operably linked to the regulatory sequence (e.g., promoter sequence) of the ADAMTS18 gene is used, an substance can be identified as inhibiting or enhancing the expression of the ADAMTS18 gene through detecting the expression level of the reporter gene product.
Illustrative reporter genes include, but are not limited to, luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa, Ade2 gene, HIS3 gene, and others well-known in the art. Methods for detection of the expression of these genes are well known in the art.
A vector containing a reporter construct may be infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on). In the context of the instant invention, the phrase "reduces the expression or activity" encompasses at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the compound, more preferably at least 25%, 50% or 75% reduction and most preferably at 95% reduction.
IV-3. Selecting therapeutic substances or agents that are appropriate for a particular individual
Differences in the genetic makeup of individuals can result in differences in their relative abilities to metabolize various drugs. An substance that is metabolized in a subject to act as an anti-tumor substance can manifest itself by inducing a change in a gene expression pattern in the subject's cells from that characteristic of a cancerous state to a gene expression pattern characteristic of a non cancerous state. Accordingly, the ADAMTS18 gene differentially expressed between cancerous and non-cancerous cells disclosed herein allow for a putative therapeutic or prophylactic inhibitor of cancer to be tested in a test cell population from a selected subject in order to determine if the substance is a suitable inhibitor of cancer in the subject.
To identify an inhibitor of cancer that is appropriate for a specific subject, a test cell population from the subject is exposed to a candidate therapeutic substance or agent, and the expression of ADAMTS18 gene is determined.
In the context of the method of the present invention, test cell populations contain cancer cells expressing the ADAMTS18 gene. Preferably, the test cell is a lung and esophageal epithelial cell.
Specifically, a test cell population may be incubated in the presence of a candidate therapeutic substance or agent and the expression of the ADAMTS18 gene in the test cell population may be measured and compared to one or more reference profiles, e.g., a cancerous reference expression profile or a non-cancerous reference expression profile.
A decrease in the expression of the ADAMTS18 gene in a test cell population relative to a reference cell population containing cancer indicates that the substance has therapeutic potential. Alternatively, a similarity in the expression of the ADAMTS18 gene in a test cell population relative to a reference cell population not containing cancer indicates that the substance has therapeutic potential.
V. Pharmaceutical compositions for treating or preventing cancer
The substances screened by any of the screening methods of the present invention, antisense nucleic acids and double-stranded molecules (e.g., siRNA) against the ADAMTS18 gene, and antibodies against the ADAMTS18 polypeptide inhibit or suppress the expression of the ADAMTS18 gene, or the biological activity of the ADAMTS18 polypeptide and inhibit or disrupt cancer cell cycle regulation, cancer cell proliferation and cell invasion. Thus, the present invention provides compositions for treating or preventing cancer, wherein the compositions include substances identified by any of the screening methods of the present invention, antisense nucleic acids or double-stranded molecules against the ADAMTS18 gene, or antibodies against the ADAMTS18 polypeptide. The present compositions can be used for treating or preventing cancer, in particular, cancer such as lung and esophageal cancer.
The compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.
In the context of the present invention, suitable pharmaceutical formulations for the active ingredients of the present invention detailed below (including screened substances, antisense nucleic acids, double-stranded molecules, antibodies, etc.) include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Preferably, administration is intravenous. The formulations are optionally packaged in discrete dosage units.
Pharmaceutical formulations suitable for oral administration include capsules, microcapsules, cachets and tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, elixirs, granules, solutions, suspensions and emulsions. The active ingredient is optionally administered as a bolus electuary or paste. Alternatively, according to needs, the pharmaceutical composition may be administered non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the active ingredients of the present invention can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending substances, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation. The amount of active ingredient contained in such a preparation makes a suitable dosage within the indicated range acquirable.
Examples of additives that can be admixed into tablets and capsules include, but are not limited to, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry. A tablet may be made by compression or molding, optionally with one or more formulational ingredients. Compressed tablets may be prepared by compressing in a suitable machine in which the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made via molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient in vivo. A package of tablets may contain one tablet to be taken on each of the month.
Furthermore, when the unit-dosage form is a capsule, a liquid carrier, such as oil, can be further included in addition to the above ingredients.
Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle prior to use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.
Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Moreover, sterile composites for injection can be formulated following normal drug implementations using vehicles, such as distilled water, suitable for injection. Physiological saline, glucose, and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injection. These can be used in conjunction with suitable solubilizers, such as alcohol, for example, ethanol; polyalcohols, such as propylene glycol and polyethylene glycol; and non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.
Sesame oil or soybean oil can be used as an oleaginous liquid, which may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer, and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and/or an anti-oxidant. A prepared injection may be filled into a suitable ampoule.
Formulations for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example, buccally or sublingually, include lozenges, which contain the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles including the active ingredient in a base such as gelatin, glycerin, sucrose or acacia. For intra-nasal administration of an active ingredient, a liquid spray or dispersible powder or in the form of drops may be used. Drops may be formulated with an aqueous or non-aqueous base also including one or more dispersing agents, solubilizing agents or suspending agents.
For administration by inhalation the compositions are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may include a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the compositions may take the form of a dry powder composition, for example, a powder mix of an active ingredient and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.
Other formulations include implantable devices and adhesive patches that release a therapeutic agent.
When desired, the above-described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
It should be understood that, in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
Preferred unit dosage formulations are those containing an effective dose, as recited under the item of 'VI. Method for treating or preventing cancer' (infra), of each active ingredients of the present invention or an appropriate fraction thereof.
V-1. Pharmaceutical compositions containing screened substances
The present invention provides compositions for treating or preventing cancers including any of the substances identified by the above-described screening methods of the present invention.
A substances identified by the method of the present invention can be directly administered or can be formulated into a dosage form according to any conventional pharmaceutical preparation method detailed above.
V-2. Pharmaceutical compositions including double-stranded molecules
Double-stranded molecules (e.g., siRNA) against the ADAMTS18 gene can be used to reduce the expression level of the genes. Herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)) as described in "Definitions". In the context of the present invention, double-stranded molecules include a sense nucleic acid sequence and an anti-sense nucleic acid sequence against the ADAMTS18 gene. The double-stranded molecule is constructed so that it includes both a portion of the sense and complementary antisense sequences of the target gene (i.e., the ADAMTS18 gene), and may also be a single construct taking a hairpin structure, wherein the sense and antisense strands are linked via a single-strand.
The double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells. The identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA. Thus, a double-stranded molecule of the present invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the ADAMTS18 gene under stringent conditions. Herein, the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule is referred to as "target sequence" or "target nucleic acid" or "target nucleotide". In the present invention, nucleotide sequence of the "target sequence" can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
In the context of the present invention, a double-stranded molecule is preferably less than 500, 200, 100, 50, or 25 base pairs in length. More preferably, a double stranded molecule is 19-25 base pairs in length. Exemplary target sequences of double-stranded molecules against the ADAMTS18 gene include the nucleotide sequences of SEQ ID NO: 11 and 12. Accordingly, for example, the present pharmaceutical composition may include a double-stranded RNA molecule (i.e., siRNA) including the nucleotide sequence 5'- GCCAGUAUCUCAAGAAAUU -3' (for SEQ ID NO: 11), and 5'- GGGCACAACUUUGGUAUGA -3' (for SEQ ID NO: 12)
as the sense strand.
In order to enhance the inhibition activity of the double-stranded molecule, 3' overhangs can be added to the 3'end of the target sequence in the sense and/or antisense strand. The number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added nucleotides form a single strand at the 3'end of the sense and/or antisense strand of the double-stranded molecule. The nucleotides to be added is preferably "u" or "t", but are not limited to.
A loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense strands in order to form a hairpin loop structure. Thus, the double-stranded molecule contained in the pharmaceutical composition of the present invention may take the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to [A]. . Herein, the polynucleotide strand which includes a sequence corresponding to a target sequence, may be referred to as "sense strand". In preferred embodiments, [A] is the sense strand; [B] is a single stranded polynucleotide consisting of 3 to 23 nucleotides; and [A'] is a polynucleotide strand which includes the antisense strand containing a complementary sequence of a target sequence, specifically hybridizing to an mRNA or a cDNA of the ADAMTS18 gene (i.e., a sequence hybridizing to the target sequence of the sense strand [A]). Herein, the polynucleotide strand which includes a complementary sequence to a target sequence, specifically hybridizing to an mRNA or a cDNA of the ADAMTS18 gene may be referred to as "antisense strand". The region [A] hybridizes to [A'], and then a loop consisting of the region [B] is formed. The loop sequence may be preferably 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from a group consisting of following sequences (www.ambion.com/techlib/tb/tb_506.html):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002, 418: 435-8.
UUCG: Lee NS et al., Nature Biotechnology 2002, 20:500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003, 100(4):1639-44.
UUCAAGAGA: Dykxhoorn DM et al., Nature Reviews Molecular Cell Biology 2003, 4:457-67.
'UUCAAGAGA ("ttcaagaga" in DNA)' is a particularly suitable loop sequence. Furthermore, loop sequence consisting of 23 nucleotides also provides an active siRNA (Jacque JM et al., Nature 2002, 418:435-8).
Exemplary hairpin siRNA suitable for the ADAMTS18 gene include:
5'-GCCAGUAUCUCAAGAAAUU -[b]-AAUUUCUUGAGAUACUGGC -3'
(target sequence of SEQ ID NO: 11);
5'-AAUUUCUUGAGAUACUGGC -[b]-GCCAGUAUCUCAAGAAAUU -3'
(target sequence of SEQ ID NO: 11) and;
5'-GGGCACAACUUUGGUAUGA-[b]- UCAUACCAAAGUUGUGCCC-3'
(target sequence of SEQ ID NO: 12);
5'-UCAUACCAAAGUUGUGCCC-[b]- GGGCACAACUUUGGUAUGA-3'
(target sequence of SEQ ID NO: 12).
Other nucleotide sequences of suitable double-stranded molecules for the present invention can be designed using an siRNA design computer program available from the Ambion website (www.ambion.com/techlib/ misc/siRNA_finder.html). The computer program selects nucleotide sequences for double-stranded molecule synthesis based on the following protocol.
Selection of Target Sites for double-stranded molecules:
1. Beginning with the AUG start codon of the object transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential target sites. Tuschl et al. Genes Cev 1999, 13(24):3191-7 don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 nucleotides) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the human genome database and eliminate from consideration any target sequences with significant homology to other coding sequences. The homology search can be performed using BLAST (Altschul SF et al., Nucleic Acids Res 1997, 25:3389-402; J Mol Biol 1990, 215:403-10.), which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/.
3. Select qualifying target sequences for synthesis. At Ambion, preferably several target sequences can be selected along the length of the gene to evaluate.
The double-stranded molecules of the present invention can be prepared using any chemical synthetic method known in the art. For example, according to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double-stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that includes the target sequence and antisense thereof is transcribed into the double-stranded molecule in vitro. In one embodiment for the annealing, the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5). Next, the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down. The annealed double-stranded polynucleotide can be purified by usually employed methods known in the art. Examples of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
The regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. The double-stranded molecules can be transcribed intracellularly by cloning ADAMTS18 gene template into a vector containing, e.g., an RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
Standard techniques are known in the art for introducing a double-stranded molecule into cells. For example, a double-stranded molecule can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. In these embodiments, the double-stranded molecules are typically modified as described below for antisense molecules. Other modifications are also available, for example, cholesterol-conjugated double-stranded molecule has shown improved pharmacological properties (Song et al., Nature Med 2003, 9:347-51). These conventionally used techniques may also be applied for the double-stranded molecules contained in the present compositions.
Alternatively, a DNA encoding the double-stranded molecule may be carried in a vector (hereinafter, also referred to as 'siRNA vector') and the double-stranded molecule may be contained in the present composition in the form of vector which enables expression of the double-stranded molecule in vivo. Such vectors may be produced, for example, by cloning a portion of the target sequence sufficient to inhibit the in vivo expression of the target gene into an expression vector having operatively-linked regulatory sequences (e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nature Biotechnology 2002, 20: 500-5). For example, an RNA molecule that is antisense to mRNA of the target gene is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of the target gene is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA). The sense and antisense strands hybridize in vivo to generate the double-stranded molecule construct for silencing the expression of the target gene. Alternatively, the sense and antisense strands may be transcribed together with the help of one promoter. In this case, the sense and antisense strands may be linked via a polynucleotide sequence to form a single-stranded construct having secondary structure, e.g., hairpin.
Thus, the present pharmaceutical composition for treating or preventing cancer may include either the double-stranded molecule (e.g., siRNA) or a vector expressing the double-stranded molecule in vivo. In particular, the present invention provides pharmaceutical compositions for treating or preventing cancer that include a double-stranded molecule that inhibits the expression of the ADAMTS18 gene, or a vector expressing the double-stranded molecule in vivo.
Further, the present invention also provides pharmaceutical compositions for inhibiting cancer cell proliferation, such composition including a double-stranded molecule which inhibits the expression of the ADAMTS18 gene, or a vector expressing the double-stranded molecule in vivo.
For introducing the double-stranded molecule vector into the cell, transfection-enhancing agent can be used. FuGENE6 (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent. Therefore, the present pharmaceutical composition may further include such transfection-enhancing agents.
In another embodiment, the present invention also provides the use of the double-stranded nucleic acid molecules of the present invention or vector encoding thereof in manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene. For example, the present invention relates to a use of double-stranded nucleic acid molecule that inhibits the expression of ADAMTS18 gene in a cell that over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 11 or 12 for manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene.
Alternatively, the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the ADAMTS18 gene.
Alternatively, the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene, wherein the method or process includes step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of ADAMTS18 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 11 or 12 as active ingredients.
In another embodiment, the present invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the ADAMTS18 gene, wherein the method or process includes step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of ADAMTS18 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 11 or 12.
V-3. Pharmaceutical compositions including antisense nucleic acids
Antisense nucleic acids targeting the ADAMTS18 gene can be used to reduce the expression level of the gene that is up-regulated in cancerous cells including lung and esophageal cancer cells. Such antisense nucleic acids are useful for the treatment of cancer, in particular lung and esophageal cancer and thus are also encompassed by the present invention. An antisense nucleic acid acts by binding to the nucleotide sequence of the ADAMTS18 gene, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the gene, promoting the degradation of the mRNAs, and/or inhibiting the expression of the protein encoded by the gene.
Thus, as a result, an antisense nucleic acid inhibits the ADAMTS18 protein to function in the cancerous cell. Herein, the phrase "antisense nucleic acids" refers to nucleotides that specifically hybridize to a target sequence and includes not only nucleotides that are entirely complementary to the target sequence but also that include mismatches of one or more nucleotides. For example, the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least 70% or higher, preferably of at least 80% or higher, more preferably of at least 90% or higher, even more preferably of at least 95% or higher over a span of at least 15 continuous nucleotides of the ADAMTS18 gene or the complementary sequence thereof. Algorithms known in the art can be used to determine such homology.
Antisense nucleic acids of the present invention act on cells that produce proteins encoded by the ADAMTS18 gene by binding to the DNA or mRNA of the gene, inhibiting their transcription or translation, promoting the degradation of the mRNA, and inhibiting the expression of the protein, finally inhibiting the protein to function.
Antisense nucleic acids of the present invention can be made into an external preparation, such as a liniment or a poultice, by admixing it with a suitable base material which is inactive against the nucleic acids.
Also, as needed, the antisense nucleic acids of the present invention can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives of these. These can be prepared by following known methods.
The antisense nucleic acids of the present invention inhibit the expression of the ADAMTS18 gene and are useful for suppressing the biological activity of the protein. In addition, expression-inhibitors, including antisense nucleic acids of the present invention, are useful in that they can inhibit the biological activity of the ADAMTS18 protein.
The antisense nucleic acids of present invention also include modified oligonucleotides. For example, thioated oligonucleotides may be used to confer nuclease resistance to an oligonucleotide.
V-4. Pharmaceutical compositions including antibodies
The function of a gene product of the ADAMTS18 gene which is over-expressed in cancers, in particular lung and esophageal cancer can be inhibited by administering a compound that binds to or otherwise inhibits the function of the gene products. An antibody against the ADAMTS18 polypeptide can be mentioned as such a compound and can be used as the active ingredient of a pharmaceutical composition for treating or preventing cancer.
The present invention relates to the use of antibodies against a protein encoded by the ADAMTS18 gene, or fragments of the antibodies. As used herein, the term "antibody" refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto. Molecules including the antigen that was used for synthesizing the antibody and molecules including the epitope of the antigen recognized by the antibody can be mentioned as closely related antigens thereto.
Furthermore, an antibody used in the present pharmaceutical compositions may be a fragment of an antibody or a modified antibody, so long as it binds to the protein encoded by the ADAMTS18 gene (e.g., an immunologically active fragment of anti- ADAMTS18 antibody). For instance, the antibody fragment may be Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston JS et al., Proc Natl Acad Sci USA 1988, 85:5879-83). Such antibody fragments may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co MS et al., J Immunol 1994, 152:2968-76; Better M et al., Methods Enzymol 1989, 178:476-96; Pluckthun A et al., Methods Enzymol 1989, 178:497-515; Lamoyi E, Methods Enzymol 1986, 121:652-63; Rousseaux J et al., Methods Enzymol 1986, 121:663-9; Bird RE et al., Trends Biotechnol 1991, 9:132-7).
An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). The present invention includes such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.
Alternatively, the antibody used for the present invention may be a chimeric antibody having a variable region derived from a non-human antibody against the ADAMTS18 polypeptide and a constant region derived from a human antibody, or a humanized antibody, including a complementarity determining region (CDR) derived from a non-human antibody, a frame work region (FR) and a constant region derived from a human antibody. Such antibodies can be prepared by using known technologies. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see e.g., Verhoeyen et al., Science 1988, 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
Complete human antibodies including human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom et al., J Mol Biol 1992, 227:381-8). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
When the obtained antibody is to be administered to the human body (antibody treatment), a human antibody or a humanized antibody is preferable for reducing immunogenicity.
Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and D Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto. A protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
Exemplary chromatography, with the exception of affinity includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
VI. Methods for treating or preventing cancer
Cancer therapies directed at specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-tumor pharmaceuticals such as trastuzumab (Herceptin) for the treatment of advanced cancers, imatinib mesylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F et al., Clin Cancer Res 2001, 7:2958-70, Review; Slamon DJ et al., N Engl J Med 2001, 344:783-92; Rehwald U et al., Blood 2003, 101:420-4; Fang G et al., Blood 2000, 96:2246-53). These drugs are clinically effective and better tolerated than traditional anti-tumor agents because they target only transformed cells. Hence, such drugs not only improve survival and quality of life for cancer patients, but also validate the concept of molecularly targeted cancer therapy. Furthermore, targeted drugs can enhance the efficacy of standard chemotherapy when used in combination with it (Gianni L, Oncology 2002, 63 Suppl 1:47-56; Klejman A et al., Oncogene 2002, 21:5868-76). Therefore, future cancer treatments will probably involve combining conventional drugs with target-specific agents aimed at different characteristics of tumor cells such as angiogenesis and invasiveness.
These modulatory methods can be performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). The methods involve administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid molecules as therapy to counteract aberrant expression of the differentially expressed genes or aberrant activity of their gene products.
Diseases and disorders characterized by increased (relative to a subject not suffering from the disease or disorder) expression levels or biological activities of genes and gene products, respectively, may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene. Therapeutics that antagonize activity can be administered therapeutically or prophylactically.
Accordingly, therapeutics that may be utilized in the context of the present invention include, e.g., (i) a polypeptide of the over-expressed ADAMTS18 gene or analogs, derivatives, fragments or homologs thereof; (ii) antibodies against the over-expressed gene or gene products; (iii) nucleic acids encoding the over-expressed gene; (iv) antisense nucleic acids or nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the nucleic acids of over-expressed gene); (v) double-stranded molecules (e.g., siRNA); or (vi) modulators (i.e., inhibitors, antagonists that alter the interaction between an over-expressed polypeptide and its binding partner). The dysfunctional antisense molecules are utilized to "knockout" endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 1989, 244: 1288 92).
Increased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered). Methods that are well known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
Therapeutic methods of the present invention may include the step of administering an agent that modulates one or more of the activities of the ADAMTS18 gene products. Examples of agent that modulate protein activity include, but are not limited to, nucleic acids, proteins, naturally occurring cognate ligands of such proteins, peptides, peptidomimetics, and other small molecule.
Thus, the present invention provides methods for treating or alleviating a symptom of cancer, or preventing cancer in a subject by decreasing the expression of the ADAMTS18 gene or the activity of the gene product. The present method is particularly suited for treating or preventing lung and esophageal cancer.
Suitable therapeutics can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing cancers. Such subjects can be identified by using standard clinical methods or by detecting an aberrant expression level ("up-regulation" or "over-expression") of the ADAMTS18 gene or aberrant activity of the gene product.
According to an aspect of the present invention, substances identified through the screening methods of the present invention may be used for treating or preventing cancer. Methods well known to those skilled in the art may be used to administer the substances to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration. If the substances are encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector can be administered to a patient to perform the therapy.
The dosage and methods for administration vary according to the body-weight, age, sex, symptom, condition of the patient to be treated and the administration method; however, one skilled in the art can routinely select suitable dosage and administration method.
For example, although the dose of a substance that binds to an ADAMTS18 polypeptide or regulates the activity of the polypeptide depends on the aforementioned various factors, the dose is generally about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult human (60 kg weight).
When administering the agent parenterally, in the form of an injection to a normal adult human (60 kg weight), although there are some differences according to the patient, target organ, symptoms and methods for administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. In the case of other animals, the appropriate dosage amount may be routinely calculated by converting to 60 kg of body-weight.
Similarly, a pharmaceutical composition of the present invention may be used for treating or preventing cancer. Methods well known to those skilled in the art may be used to administer the compositions to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration.
For each of the aforementioned conditions, the compositions, e.g., polypeptides and organic compounds, can be administered orally or via injection at a dose ranging from about 0.1 to about 250 mg/kg per day. The dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day. Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.
The dose employed will depend upon a number of factors, including the age, body weight and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity. In any event, appropriate and optimum dosages may be routinely calculated by those skilled in the art, taking into consideration the above-mentioned factors.
In particular, an antisense nucleic acid against the ADAMTS18 gene can be given to the patient by direct application onto the ailing site or by injection into a blood vessel so that it will reach the site of ailment.
The dosage of the antisense nucleic acid derivatives of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
In the preferred embodiments, the method of the present invention includes the step of administering the double-stranded molecule against the ADAMTS18 gene to a subject. The preferred examples of the double-stranded molecule to be administered are described under the item of " V-2. Pharmaceutical compositions including double-stranded molecules" and the following item of "VII. Double-stranded molecules and vectors encoding them"
It is understood that the double-stranded molecules of the present invention degrade the mRNA of the ADAMTS18 gene in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.
One skilled in the art can readily determine an effective amount of the double-stranded molecule of the present invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered. The precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
The present methods can be used to inhibit the growth or metastasis of cancer; for example lung cancer, especially lung cancer and esophageal cancer. For treating cancer, the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule. Alternatively, the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer. For example, the double-stranded molecule of the present invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents).
In the context of the present methods, the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector that expresses the double-stranded molecule.
Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes. A preferred delivery reagent is a liposome.
Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule. Liposomes suitable for use in the context of the present invention may be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
Preferably, the liposomes encapsulating the present double-stranded molecule includes a ligand molecule that can deliver the liposome to the cancer site. Ligands which bind to receptors prevalent in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.
Particularly preferably, the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure. In one embodiment, a liposome of the invention can include both opsonization-inhibition moieties and a ligand.
Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature. Thus, target tissue characterized by such microvasculature defects, for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen. Thus, liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
The opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
Vectors expressing a double-stranded molecule of the present invention are discussed in the following item. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for delivering recombinant viral vectors, which express a double-stranded molecule of the invention, to an area of cancer in a patient are within the skill of the art.
The double-stranded molecule of the present invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites. For example, the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
Suitable enteral administration routes include oral, rectal, or intranasal delivery.
Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of the cancer.
The double-stranded molecule of the present invention can be administered in a single dose or in multiple doses. Where the administration of the double-stranded molecule of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the agent directly into the tissue is at or near the site of cancer preferred. Multiple injections of the agent into the tissue at or near the site of cancer are particularly preferred.
One skilled in the art can also readily determine an appropriate dosage regimen for administering the double-stranded molecule of the invention to a given subject. For example, the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days. In a preferred dosage regimen, the double-stranded molecule is injected at or near the site of cancer once a day for seven days. Where a dosage regimen includes multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can include the total amount of a double-stranded molecule administered over the entire dosage regimen.
In the present invention, a cancer overexpressing ADAMTS18 can be treated with at least one active ingredient selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof.
Examples of cancer to be treated include, but are not limited to, lung and esophageal cancer. Accordingly, prior to the administration of a double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of ADAMTS18 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ. Thus, in one embodiment, the present invention provides a method for treating a cancer (over)expressing ADAMTS18, which method may include the steps of:
i) determining the expression level of ADAMTS18 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated;
ii) comparing the expression level of ADAMTS18 with normal control; and
iii) administrating at least one component selected from the group consisting of
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
to a subject with a cancer overexpressing ADAMTS18 compared with normal control. Alternatively, the present invention also provides a pharmaceutical composition including at least one component selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
for use in administrating to a subject having a cancer overexpressing ADAMTS18. In other words, the present invention further provides a method for identifying a subject to be treated with:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof,
which method may include the step of determining an expression level of ADAMTS18 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with a double-stranded molecule of the present invention.
The method of treating a cancer of the present invention will be described in more detail below.
A subject to be treated by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
According to the present invention, the expression level of ADAMTS18 in cancer cells or tissues obtained from a subject is determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, hybridization methods (e.g., Northern hybridization), a chip or an array, probes, RT-PCR can be used to determine the transcription product level of ADAMTS18.
Alternatively, the translation product may be detected for the treatment of the present invention. For example, the quantity of observed protein (SEQ ID NO: 2) may be determined.
As another method to detect the expression level of ADAMTS18 gene based on its translation product, the intensity of staining may be measured via immunohistochemical analysis using an antibody against the ADAMTS18 protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of ADAMTS18 gene.
Methods for detecting or measuring the ADAMTS18 polypeptide and/or polynucleotide encoding thereof can be exemplified as described above (I. Diagnosing cancer).
VII. Double-stranded molecules and vectors encoding them
Herein, an siRNA including either of the sequences of SEQ ID NOs: 11 or 12 was demonstrated to suppress cell growth or viability of cells expressing the ADAMTS18 gene. Therefore, double-stranded molecules including any of these sequences and vectors expressing the molecules are considered to serve as preferable pharmaceutics for treating or preventing diseases which involve the proliferation of ADAMTS18 gene expressing cells, for example, cancer, particularly lung and esophageal cancer. Thus, according to an aspect, the present invention provides double-stranded molecules including the target sequence selected from the group consisting of SEQ ID NOs: 11 and 12 and vectors expressing the molecules. More specifically, the present invention provides a double-stranded molecule, when introduced into a cell expressing the ADAMTS18 gene, inhibits expression of the gene, wherein the double-stranded molecule includes a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 and 12 as a target sequence, and the antisense strand includes a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule. Alternatively, the present invention provides a double-stranded molecule, when introduced into a cell expressing an ADAMTS18 gene, inhibits expression of the gene, wherein the double-stranded molecule has a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 11 and 12, and the antisense strand has a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
The target sequence for the ADAMTS18 gene included in the sense strand may consist of a sequence of a portion of SEQ ID NO: 1 that is less than about 500, 400, 300, 200, 100, 75, 50 or 25 contiguous nucleotides. Preferably, the target sequence may be from about 19 to about 25 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1. The present invention is not limited thereto, but suitable target sequences include the nucleotide sequences selected from the group consisting of SEQ ID NOs: 11 and 12.
The double-stranded molecule of the present invention may be composed of two polynucleotide constructs, i.e., a polynucleotide including the sense strand and a polynucleotide including the antisense strand. Alternatively, the molecule may be composed of one polynucleotide construct; i.e., a polynucleotide including both the sense strand and the antisense strand, wherein the sense and antisense strands are linked via a single-stranded polynucleotide which enables hybridization of the target sequences within the sense and antisense strands by forming a hairpin structure. Herein, the single-stranded polynucleotide may also be referred to as "loop sequence" or "single-strand". The single-stranded polynucleotide linking the sense and antisense strands may consist of 3 to 23 nucleotides. See under the item of "V-2. Pharmaceutical compositions including double-stranded molecules" for more details on the double-stranded molecule of the present invention.
The double-stranded molecules of the present invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. The skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; WO2005/045037). In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).
In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212). In another embodiment, modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents such as US20060234970 are available. The present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
Furthermore, the double-stranded molecules of the invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule including both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule. The hybrid of a DNA strand and an RNA strand may be the hybrid in which either the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may be either the molecule that both of the sense and antisense strands are composed of DNA and RNA, or the molecule that any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
In order to enhance stability of the double-stranded molecule, the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression. As a preferred example of the chimera type double-stranded molecule, an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of the double-stranded molecule is RNA. The upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. That is, in preferable embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA. For instance, the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
sense strand:
5'-[---DNA---]-3'
3'-(RNA)-[DNA]-5'
:antisense strand,
sense strand:
5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5'
:antisense strand, and
sense strand:
5'-(RNA)-[DNA]-3'
3'-(---RNA---)-5'
:antisense strand.
The upstream partial region preferably is a domain consisting of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules. Moreover, preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
In the context of the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
Alternatively, the present invention provides vectors including each of a combination of polynucleotide having a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid includes nucleotide sequence of SEQ ID NOs: 11 or 12, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the ADAMTS18, inhibit expression of said gene. Preferably, the sense strand of the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1). More preferably, the combination of polynucleotide includes a single nucleotide transcript having the sense strand and the antisense strand linked via a single-stranded nucleotide sequence. More preferably, the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is a nucleotide sequence including SEQ ID NO: 11 or 12; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to [A].
Vectors of the present invention can be produced, for example, by cloning ADAMTS18 sequence into an expression vector so that regulatory sequences are operatively-linked to ADAMTS18 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5). For example, RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA). The sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene. Alternatively, two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and anti-sense strands and then forming a double-stranded molecule construct. Furthermore, the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
The vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).
The vectors of the present invention include, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell. Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
Hereinafter, the present invention is described in more detail with reference to the Examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Materials and Methods
Cell lines and tissue samples.
The 15 human lung-cancer cell lines used in this example included: five adenocarcinomas (ADCs; NCI-H1781, NCI-H1373, LC319, A549 and PC-14), five squamous-cell carcinomas (SCCs; SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170 and LU61), one large-cell carcinoma (LCC; LX1), and four small-cell lung cancers (SCLCs; SBC-3, SBC-5, DMS114 and DMS273). The 10 human esophageal carcinoma cell lines used in this study were as follows; 9 SCC cell lines (TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, and TE10) and one ADC cell line (TE7) (Nishihira T, et al. J Cancer Res Clin Oncol 1993;119:441-9.). All cells were grown in monolayer in appropriate media supplemented with 10% fetal calf serum (FCS) and were maintained at 37 degrees C in humidified air with 5% CO2. Human small airway epithelial cells (SAEC) used as a normal control were grown in optimized medium (SAGM) from Cambrex Bio Science Inc. Primary lung cancer and ESCC samples had been obtained earlier with informed consent (Kikuchi T, Daigo Y, Katagiri T, et al. Oncogene 2003;22:2192-205, Taniwaki M, Daigo Y, Ishikawa N, et al. Int J Oncol 2006;29:567-75, Yamabuki T, Daigo Y, Kato T, et al. Int J Oncol 2006;28:1375-84).
Semiquantitative RT-PCR.
A total of 3 microgram aliquot of mRNA from each sample was reversely transcribed to single-stranded cDNAs using random primer (Roche Diagnostics) and Superscript II (Invitrogen). Semiquantitative RT-PCR experiments were carried out with the following sets of synthesized primers specific for human epithelial cell transforming sequence 2 (ADAMTS18) or with beta-actin (ACTB)-specific primers as an internal control: ADAMTS18, 5'-GGATTAGCCAGCTCAGCATA-3' (SEQ ID NO: 3) and 5'-CTGTTTTTCAGAAGGCAACG-3' (SEQ ID NO: 4); ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3' (SEQ ID NO: 5) and 5'-CAAGTCAGTGTACAGGTAAGC-3' (SEQ ID NO: 6). PCR reactions were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification.
Northern-blot analysis.
Human multiple-tissue blots covering 23 tissues (BD Bioscience) were hybridized with an [alpha-32P]-dCTP-labeled, 226-bp PCR product of ADAMTS18 that was prepared as a probe using primers 5'-TAGGGCAACATGGACTGTTTAAG-3' (SEQ ID NO: 7) and 5'- GCTGTGTTTTGTCATTTAGCTCC-3' (SEQ ID NO: 8). Prehybridization, hybridization, and washing were performed following manufacturer's specifications. The blots were autoradiographed with intensifying screens at -80 degrees C for 7 days.
RNA interference assay.
To evaluate the biological functions of ADAMTS18 in cancer cells, a short-hairpin RNA against the target genes were used. The target sequences of the synthetic oligonucleotides for RNAi were as follows: control 1 (enhanced green fluorescent protein gene (EGFP), a mutant of Aequorea victoria GFP), 5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID NO: 9); control 2 (Luciferase (LUC): Photinus pyralis luciferase gene), 5'-CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 10); si-ADAMTS18-#1, 5'-GCCAGUAUCUCAAGAAAUU-3' (SEQ ID NO: 11); si-ADAMTS18-#2, and 5'-GGGCACAACUUUGGUAUGA-3' (SEQ ID NO: 12). DMS114 and TE4 cells were plated onto 10-cm dishes (1.5x 106 cells per dish), and transfected with either of the siRNA oligonucleotides (100nM), using 24 microliter of Lipofectamine 2000 (Invitrogen), according to the manufacturers' instructions. After 7 days of incubation, these cells were stained by Giemsa solution to assess colony formation, and cell numbers were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Preparation of ADAMTS18 expression plasmids.
To investigate the biological function of ADAMTS18, p3XFLAG-tagged (C-terminal) plasmids expressing full-length fragments of ADAMTS18 were prepared.
Western-blotting.
Cells were lysed in lysis buffer; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and Protease Inhibitor Cocktail Set III (Calbiochem). The protein content of each lysate was determined by a Bio-Rad protein assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. Ten micrograms of each lysate were resolved on 5-15% denaturing polyacrylamide gels (with 4% polyacrylamide stacking gel) and transferred electrophoretically to a nitrocellulose membrane (GE Healthcare Bio-sciences). After blocking with 5% non-fat dry milk in TBST, the membrane was incubated with ANTI-FLAG, antibody produced in rabbit (SIGMA) for 1 hour at room temperature. Immunoreactive proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 hour at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-sciences). When cell culture media was applied for western-blotting, supernatant was collected after centrifugation for 10-15 minutes at 1,000X g, 4 degrees C. Then, supernatant were concentrated by using Amicon Ultra-15 Centrifugal Filter Devices (MILLIPORE), according to the manufacturers' instructions.
Cell growth assay.
COS-7 and A549 cells transfected either with p3XFLAG-tagged (C-terminal) plasmids expressing ADAMTS18 or with mock plasmids were seeded on to 6-well microtiter plates (1 x 104 cells/well). After 7 days of incubation, these cells were evaluated using Cell Counting Kits as directed by the supplier (Wako).
N-glycosidase assay.
COS-7 cells transfected either with p3XFLAG-tagged (C-terminal) plasmids expressing ADAMTS18 or with mock plasmids were harvested as described above, and each samples were mixed with N-Glycosidase F (CALBIOCHEM). After incubation at 37 degrees C for 16 hours, Western-blotting analysis was performed.
Immunocytochemical analysis.
Cells were plated on glass coverslips (Becton Dickinson Labware), fixed with 4% paraformaldehyde, and permeablilized with 0.1% Triton X-100 in PBS for 3 minutes at room temperature. Non-specific binding was blocked by CASBLOCK (ZYMED) for 10 minutes at room temperature. Cells were then incubated for 60 minutes at room temperature with ANTI-FLAG, antibody produced in rabbit (SIGMA) diluted in PBS containing 1% BSA. After being washed with PBS, the cells were stained by FITC-conjugated secondary antibody (SantaCruz) for 60 minutes at room temperature. After an additional wash with PBS, each specimen was mounted with Vectashield (Vector Laboratories) containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS, Leica Microsystems).
Matrigel invasion assay.
COS-7 cells transfected either with p3XFLAG-tagged (C-terminal) plasmids expressing ADAMTS18 or with mock plasmids were grown to near confluence in DMEM containing 10% FCS. The cells were harvested by trypsinization, washed in DMEM without addition of serum or proteinase inhibitor, and suspended in DMEM at concentration of 1 x 105 cells/ml. Before preparing the cell suspension, the dried layer of Matrigel matrix (Becton Dickinson Labware) was rehydrated with DMEM for 2 hours at room temperature. DMEM (0.75 ml) containing 10% FCS was added to each lower chamber in 24-well Matrigel invasion chambers, and 0.5 ml (5 x 104 cells) of cell suspension was added to each insert of the upper chamber. The plates of inserts were incubated for 24 hours at 37 degrees C. After incubation, the chambers were processed; cells invading through the Matrigel were fixed and stained by Giemsa as directed by the supplier (Becton Dickinson Labware). At same time point, cell growth assay was examined. Next, DMS114 cells transfected with either si-ADMTS18-#1 or control1 (EGFP) were seeded in Matrigel invasion chambers after 24 hours of transfection and examined cellular invasiveness as like as above.
Matrix metalloproteinase assay.
Two types of cells were prepared; COS-7 cells transfected either with p3XFLAG-tagged (C-terminal) plasmids expressing ADAMTS18 or with mock plasmids were grown to near confluence in DMEM containing 10% FCS, whereas DMS114 cells transfected either with si-ADAMTS18-#1 or with control 1 (EGFP) were incubated for 48 hours. Then, supernatant of cell culture media were collected and centrifuged for 10-15 minutes at 1,000X g, at 4 degrees C. The supernatant were collected and examined for Matrix Metalloproteinase (MMP) activity, using SensoLyte 570 Generic Assay Kit (AnaSpec), according to the manufacturers' instructions. The MMP containing-samples were incubated with 1 mM 4-aminophenyl mercuric acetate for 2 hours to activate MMP. The samples and generic MMP substrate (containing substrates of MMPs-1, 2, 7, 8, 9, 10, 13 and 14) were mixed to start enzymatic reaction and incubated at room temperature for 30 minutes. Fluorescence intensity was measured by Microplate reader.
Results
ADAMTS18 expression in lung and esophageal cancers and normal tissues.
The genome-wide expression profile analysis was carried out for 101 lung carcinomas (86 NSCLCs and 15 small-cell lung cancers) and 19 ESCCs, as well as 30 normal organs, using cDNA microarray consisting of 27,648 genes or expressed sequence tags (Kikuchi T, Daigo Y, Katagiri T, et al. Oncogene 2003;22:2192-205, Kakiuchi S, Daigo Y, Tsunoda T, et al. Mol Cancer Res 2003;1:485-99, Kakiuchi S, Daigo Y, Ishikawa N, et al. Hum Mol Genet 2004;13:3029-43, Kikuchi T, Daigo Y, Ishikawa N, et al. Int J Oncol 2006; 28:799-805, Taniwaki M, Daigo Y, Ishikawa N, et al. Int J Oncol 2006;29:567-75, Yamabuki T, Daigo Y, Kato T, et al. Int J Oncol 2006;28:1375-84). The elevated expression (>=3-fold) of ADAMTS18 transcript was identified in cancer cells in the great majority of the lung and esophageal cancer samples examined. Moreover, no ADAMTS18 expression was observed in any of 29 normal tissues except testis (data not shown). Therefore, ADAMTS18 was considered to be a good molecular candidate for further analyses. Its overexpression was confirmed by semiquantitative RT-PCR experiments in 7 of 15 lung cancer tissues, in 2 of 15 lung-cancer cell lines, in 5 of 10 ESCC tissues, and in 1 of 10 ESCC cell lines examined (Figs. 1A and 1B). In 5 ESCC patients, the ADAMTS18 expression between ESCC tissues and their normal esophagus tissues was also compared (Figs. 1C). Northern blot analysis using an ADAMTS18 cDNA as a probe identified a transcript of 6.0-kb only in placenta among 23 normal human tissues examined (Fig. 1D).
Inhibition of growth of cancer cells by small interfering RNA for ADAMTS18.
To assess whether ADAMTS18 is essential for growth or survival of lung and esophageal cancer cells, synthetic oligonucleotide siRNAs against ADAMTS18 was used and transfected into DMS114 and TE4 cells that endogenously expressed high levels of ADAMTS18. The ADAMTS18-mRNA levels in cells transfected with si-ADAMTS18-#1 or -#2 were significantly decreased in comparison with cells transfected with either control siRNAs (Fig. 2, top panels). MTT assays and colony-formation assays revealed a drastic reduction in the number of cells transfected with si-ADAMTS18-#1 or -#2 (Fig. 2, middle and bottom panels).
Effect of exogenous ADAMTS18 on cell growth.
To assess a potential role of ADAMTS18 in tumorigenesis, plasmids designed to express ADAMTS18 (pCAGGSn3FC-ADAMTS18) was prepared and transfected into COS-7 cells. After confirmation of ADAMTS18 expression by western-blotting (Fig. 3A, left panels), MTT assays was carried out, and it was found that growth of the COS-7-ADAMTS18 cells was promoted at a significant degree in comparison to the COS-7 cells transfected with the mock vector (Fig. 3A, right panel). Next, A549 cells were transfected as same as COS-7 cells, and it revealed that exogenous ADAMTS18 promoted cell growth of A549 (Figs. 3B).
Post-translational modification of ADAMTS18.
To examine a post-translational modification of exogenous ADAMTS18, N-glycosidase assay was performed. After incubation with N-glycosidase, western-blot analysis revealed that the band of exogenous ADAMTS18 protein was shifted lower (Fig. 4A). The results suggested that ADAMTS18 could be N-glycosylated after translation.
Subcellular localization of exogenous ADAMTS18 protein.
To examine the subcellular localization of exogenous ADAMTS18 protein, immunocytochemical analysis was performed by using ANTI-FLAG. Exogenous ADAMTS18 protein, at 72-hour after transfection, located in the cytoplasm of COS-7 cells (Fig. 4B). Localization of ADAMTS18 protein looked like granulous pattern. It might be secretion vesicles.
Secretion of exogenous ADAMTS18 protein into culture medium.
ADAMTS18 was supposed to be a secreted protein, so the secretion of ADAMTS18 protein into culture medium was evaluated by using COS-7 cells, which were not or very lower expressed ADAMTS18 (Fig. 4C), overexpressed ADAMTS18. At 48-hour after transfection of ADAMTS18, culture medium was changed. Then, at 72-hour after transfection, culture medium was harvested. After centrifugation, culture medium was concentrated and applied for western-blotting analysis. Exogenous ADAMTS18 protein was appeared to be secreted into culture medium (Fig. 4D).
Activation of mammalian cellular invasion by ADAMTS18.
Because of ADAMTS18 contains metalloproteinase domain, which digests ECM (extra cellular matrix), a possible role of ADAMTS18 in cellular invasion was examined by Matrigel assays using a mammalian cells (COS-7). Transfection of ADAMTS18 cDNA into the cells significantly enhanced their invasive activity through Matrigel, compared to cells transfected with mock vector (Fig. 5A). And cell growth assays, performed at the same time point with Matrigel assays, revealed no significant differences between them (Fig. 5B). This result independently suggested that ADAMTS18 could contribute to the highly malignant phenotype of cancer cells.
MMP activity of ADAMTS18.
Since ADAMTS18 contains metalloproteinase domain and has cellular invasive effect, MMP activity of ADAMTS18 was evaluated by MMP assay. COS-7 cells which had been transfected with ADAMTS18-expressing plasmids significantly enhanced MMP activity, compared to cells transfected with mock vector (Fig. 5C). In contrast, lung cancer cells (DMS114), which had been transfected with si-ADAMTS18-#1, significantly showed decreased MMP activity in comparison with cells transfected with control siRNA (EGFP) (Fig. 5D). These results suggested that ADAMTS18 could possess the MMP activity and contribute to the invasion of cancer cells.
Discussion
A genome-wide expression profile analysis was performed for 101 lung cancers and 19 ESCCs after enrichment of cancer cells by laser microdissection, using a cDNA microarray containing 27,648 genes. As a result of the analyses, a number of genes that could be potentially good candidates for the development of novel diagnostic markers, therapeutic drugs, and/or immunotherapy was identified, and it was found that ADAMTS18 was frequently transactivated in the majority of lung and esophageal cancer samples, and that its gene products play indispensable roles in the growth and progression of the cancer cells.
The ADAM family of proteins, an MMP-related metalloproteinase family, are multifunctional proteins involved in the proteolytic processing of other transmembrane proteins, cell adhesion and cell signaling events (Mochizuki S and Okada Y. Cancer Sci 2007;98:621-8). Many transmembrane proteins are processed by one or several proteolytic steps to the biologically active configuration. Examples include growth factors such as EGF, HB-EGF and TGF-alpha, and cytokines such as TNF-alpha, all of which are synthesized as precursors. In addition, there are a number of cell surface receptors that undergo cleavage near the transmembrane domain, a process called ectodomain shedding. These include TNF-alpha receptor-I, TNF-alpha receptor-II, CD44, L-selectin and Erb4/HER4. The soluble, released ectodomains of the receptors may be part of the down-modulation in response to ligand activation, or they may have a function of their own. It has become clear over the past few years that ADAMs play a major role in these processes.
Key features of malignant tumors are their abilities to invade surrounding tissues, to have access to the vascular and lymphatic systems, and to disseminate to distant organs by metastatic spreading. Accumulating evidence demonstrates the crucial role of proteolytic enzymes such as matrix metalloproteinases (MMPs) and closely related ADAMs (a disintegrin and metalloproteinase) and ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) in cancer development and progression. Although information about functions of ADAMs and ADAMTSs in cancers is still limited, recent studies have provided evidence of dysregulation of various ADAMs and ADAMTSs in different types of cancers. Therefore, these proteins have attracted attention of many research groups and functional analysis of ADAMs and ADAMTSs are ongoing based on the recent generation of mice deficient for some of these proteins.
In the present invention, ADAMTS18 was characterized as a novel oncoprotein involved in lung and esophageal cancers. The importance of ADAMTS18 was evaluated for cancer cell growth and/or survival. The treatment of two cancer cells with specific siRNA for suppression of ADAMTS18 expression resulted in inhibition of cancer-cell growth. Additional evidences supporting the significance of ADAMTS18 in carcinogenesis were also obtained. The induction of exogenous ADAMTS18 into mammalian COS-7 cells and A549, lung cancer cells, resulted in the significant promotion of cell growth. Furthermore, induction of exogenous ADAMTS18 into COS-7 cells activated cellular invasion. As expected, ADAMTS18 was secreted into culture medium and might have MMP activity. These results strongly suggested that ADAMTS18 was likely to play important roles for cancer cell growth and invasion, and could lead to tumor development and metastasis. Because specific ADAMs have been shown to promote cancer initiation and progression, one can reasonable predict that blocking their actions would slow or prevent tumor progression. In recent years, a number of selective synthetic inhibitors against a small number of ADAMs have been described (Duffy MJ, McKiernan E, O'Donovan N, and McGowan PM. Clin Cancer Res 2009;15:1140-4). Since ADAMTS18 is properly classified as one of typical cancer-placenta antigens, selective inhibition of ADAMTS18 enzymatic activity by small molecule compounds constitutes a promising therapeutic strategy that is expected to have a powerful biological activity against cancer with a minimal risk of adverse events.
In conclusion, the data herein enable the design of new anti-cancer drugs to specifically target the oncogenic activity of ADAMTS18 for treatment of cancer patients. ADAMTS18 was identified as a novel potential therapeutic target for the patients with lung and esophageal cancers.
The gene-expression analysis of cancers described herein using the genome-wide cDNA microarray has identified specific genes as a target for cancer prevention and therapy. Based on the differentially expression of ADAMTS18 gene, the present invention provides a molecular diagnostic marker for diagnosing or detecting cancer, in particular, lung and esophageal cancer.
The data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of a molecular target for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
As demonstrated herein, cell growth is suppressed by double-stranded molecules that specifically target the ADAMTS18 gene. Thus, these novel double-stranded molecules find real-world utility as anti-cancer pharmaceuticals.
The expression of the ADAMTS18 gene is markedly elevated in cancer, specifically lung cancer and esophageal cancer, as compared to normal organs. Accordingly, this gene finds convenient, real-world utility as a diagnostic marker for cancer, in particular, lung cancer and esophageal cancer, and the proteins encoded thereby find utility in diagnostic assays for cancer.
Furthermore, the methods described herein find utility in diagnosis of cancer, including lung cancer and esophageal cancer.
Moreover, the present invention provides new therapeutic approaches for treating cancer including lung cancer and esophageal cancer. The ADAMTS18 gene finds real-world utility as a useful target for the development of anti-cancer pharmaceuticals.
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
Furthermore, while the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims (26)

  1. A method for detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of an ADAMTS18 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates that said subject suffers from or is at a risk of developing cancer, wherein said expression level is determined by any method selected from a group consisting of:
    (a) detecting mRNA of an ADAMTS18 gene;
    (b) detecting a protein encoded by an ADAMTS18 gene; and
    (c) detecting a biological activity of a protein encoded by an ADAMTS18 gene.
  2. The method of claim 1, wherein said expression level is at least 10% greater than the normal control level.
  3. The method of claim 1 or 2, wherein the biological activity is cell proliferative activity, N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity.
  4. A method of screening a candidate substance for treating or preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof;
    (b) detecting binding between the polypeptide or fragment and the test substance; and
    (c) selecting the test substance that binds to the polypeptide or fragment as a candidate substance for treating or preventing cancer.
  5. A method of screening a candidate substance for treating or preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with an ADAMTS18 polypeptide or a fragment thereof;
    (b) detecting a biological activity of the polypeptide or fragment;
    (c) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the substance; and
    (d) selecting the test substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer.
  6. The method of claim 5, wherein the biological activity is cell proliferative activity, N-glycosylation activity, invasive activity or Matrix Metalloproteinase (MMP) activity.
  7. A method of screening a candidate substance for treating or preventing cancer, which comprises steps of:
    (a) contacting a test substance with a cell expressing an ADAMTS18 gene;
    (b) detecting expression level of the ADAMTS18 gene;
    (c) comparing the expression level with the expression level detected in the absence of the test substance; and
    (d) selecting the test substance that reduces the expression level as a candidate substance for treating or preventing cancer.
  8. A method of screening a candidate substance for treating or preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with a cell introduced with a vector that comprises a transcriptional regulatory region of an ADAMTS18 gene and a reporter gene expressed under control of the transcriptional regulatory region;
    (b) measuring an expression level or activity of said reporter gene;
    (c) comparing the expression level or activity with the expression level or activity detected in the absence of the test substance; and
    (d) selecting the test substance that reduces the expression level or activity as a candidate substance for treating or preventing cancer.
  9. A double-stranded molecule, when introduced into a cell expressing an ADAMTS18 gene, inhibits expression of the gene, wherein the double-stranded molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 11 and 12, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  10. The double-stranded molecule of claim 9, wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
  11. The double-stranded molecule of claim 9 or 10, wherein said double-stranded molecule is a single polynucleotide construct comprising the sense strand and the antisense strand linked via a single-strand.
  12. The double-stranded molecule of claim 11, which has a general formula 5'-[A]-[B]-[A']-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 11 and 12, [B] is a single-strand and consists of 3 to 23 nucleotides, and [A'] is an antisense strand comprising a nucleotide sequence complementary to the target sequence selected from SEQ ID NO: 11 and 12.
  13. A vector encoding the double-stranded molecule of any one of claims 9 to 12.
  14. Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises a nucleotide sequence corresponding to SEQ ID NO: 11 or 12, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing ADAMTS18 gene, inhibit the cell proliferation.
  15. A method of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against an ADAMTS18 gene or a vector encoding said double-stranded molecule, wherein the double-stranded molecule inhibits the expression of the ADAMTS18 gene.
  16. The method of claim 15, wherein the double-stranded molecule is that of any one of claims 9 to 12.
  17. The method of claim 15, wherein the vector is that of claim 13 or 14.
  18. The method of any one of claims 1 to 8, and 15 to 17, wherein the cancer is selected from the group consisting of lung cancer and esophagus cancer.
  19. A composition for treating or preventing cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against an ADAMTS18 gene or a vector comprising said double-stranded molecule, wherein the double-stranded molecule inhibits the expression of the ADAMTS18 gene, and a pharmaceutically acceptable carrier.
  20. The composition of claim 19, wherein the double-stranded molecule is that of any one of claims 9 to 12.
  21. The composition of claim 19, wherein the vector is that of claim 13 or 14.
  22. The composition of any one of claims 19 to 21, wherein the cancer is selected from the group consisting of lung cancer and esophagus cancer.
  23. A kit for diagnosing or detecting cancer, comprising a reagent for detecting a transcription or translation product of an ADAMTS18 gene.
  24. The kit of claim 23, wherein the reagent comprises a nucleic acid that binds to a transcription product of ADAMTS18 gene or an antibody that binds to a translation product of an ADAMTS18 gene.
  25. A reagent for diagnosing or detecting cancer, comprising a nucleic acid that binds to a transcription product of ADAMTS18 gene or an antibody that binds to a translation product of an ADAMTS18 gene.
  26. The kit of claim 23 or 24, or the reagent of claim 25, wherein the cancer is selected from the group consisting of lung cancer and esophagus cancer.
PCT/JP2010/005185 2009-08-24 2010-08-23 Lung and esophageal cancer related gene adamts18 WO2011024433A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP10811492A EP2470651A1 (en) 2009-08-24 2010-08-23 Lung and esophageal cancer related gene adamts18
JP2012510050A JP2013502203A (en) 2009-08-24 2010-08-23 Lung cancer and esophageal cancer related gene ADAMTS18
CN2010800480314A CN102597235A (en) 2009-08-24 2010-08-23 Lung and esophageal cancer related gene ADAMTS18

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27503909P 2009-08-24 2009-08-24
US61/275,039 2009-08-24

Publications (1)

Publication Number Publication Date
WO2011024433A1 true WO2011024433A1 (en) 2011-03-03

Family

ID=43627555

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/005185 WO2011024433A1 (en) 2009-08-24 2010-08-23 Lung and esophageal cancer related gene adamts18

Country Status (4)

Country Link
EP (1) EP2470651A1 (en)
JP (1) JP2013502203A (en)
CN (1) CN102597235A (en)
WO (1) WO2011024433A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2883628T3 (en) * 2014-11-17 2021-12-09 Univ Queensland Glycoprotein biomarkers for adenocarcinoma of the esophagus and Barrett's esophagus and their uses

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007013665A2 (en) * 2005-07-27 2007-02-01 Oncotherapy Science, Inc. Method of diagnosing small cell lung cancer
WO2008021288A2 (en) * 2006-08-11 2008-02-21 Johns Hopkins University Consensus coding sequences of human breast and colorectal cancers
JP2008178362A (en) * 2007-01-25 2008-08-07 Hiroshima Univ Novel method for detecting cancer, cancer-detecting tool and cancer-detection kit

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101070538B (en) * 2005-03-28 2011-02-09 中国科学院广州生物医药与健康研究院 Human epiterm growth-factor receptor mutation gene and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007013665A2 (en) * 2005-07-27 2007-02-01 Oncotherapy Science, Inc. Method of diagnosing small cell lung cancer
WO2008021288A2 (en) * 2006-08-11 2008-02-21 Johns Hopkins University Consensus coding sequences of human breast and colorectal cancers
JP2008178362A (en) * 2007-01-25 2008-08-07 Hiroshima Univ Novel method for detecting cancer, cancer-detecting tool and cancer-detection kit

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIRATA D. ET AL: "Involvement of Epithelial Cell Transforming Sequence-2 Oncoantigen in Lung and Esophageal Cancer Progression", CLIN.CANCER RES., vol. 15, no. 1, 1 January 2009 (2009-01-01), pages 256 - 266, XP055004125 *
LOPEZ-OTIN C. ET AL: "Emerging roles of proteases in tumour suppression", NAT.REV.CANCER, vol. 7, no. 10, 2007, pages 800 - 808, XP008154182 *

Also Published As

Publication number Publication date
EP2470651A1 (en) 2012-07-04
CN102597235A (en) 2012-07-18
JP2013502203A (en) 2013-01-24

Similar Documents

Publication Publication Date Title
US8420329B2 (en) Methods for diagnosing or treating prostate cancer
WO2010067487A1 (en) Nectin-4 for target genes of cancer therapy and diagnosis
WO2012144220A1 (en) Ezh2 as target gene for cancer therapy and diagnosis
WO2011096211A1 (en) Whsc1 and whsc1l1 for target genes of cancer therapy and diagnosis
WO2011096210A1 (en) Prmt1 and prmt6 for target genes of cancer therapy and diagnosis
US8512944B2 (en) PRMT1 for target genes of cancer therapy and diagnosis
WO2012090479A1 (en) Mcm7 as a target gene for cancer therapy and diagnosis
WO2011161960A1 (en) C1orf59 for target genes of cancer therapy and diagnosis
US20110262463A1 (en) Syngr4 for target genes of cancer therapy and diagnosis
WO2010023837A1 (en) Breast cancer related gene rqcd1
WO2011024433A1 (en) Lung and esophageal cancer related gene adamts18
US20110160288A1 (en) Oip5 as a target gene for cancer therapy and diagnosis
US20110313018A1 (en) Cancer related gene, lgn/gpsm2
WO2012153492A1 (en) Nptx2 as tumor marker and therapeutic target for cancer
WO2011024428A1 (en) Breast cancer related gene c12orf32
WO2011021386A1 (en) Cstf2 for target genes of lung cancer therapy and diagnosis
WO2011018898A1 (en) Cdc45l as tumor marker and therapeutic target for cancer
US20130203625A1 (en) Suv420h1 and suv420h2 as target genes for cancer therapy and diagnosis

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201080048031.4

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10811492

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2012510050

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2010811492

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2604/CHENP/2012

Country of ref document: IN