US20060040883A1 - Methods for treating cancer using anti-Wnt2 monoclonal antibodies and siRNA - Google Patents

Methods for treating cancer using anti-Wnt2 monoclonal antibodies and siRNA Download PDF

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US20060040883A1
US20060040883A1 US11/131,425 US13142505A US2006040883A1 US 20060040883 A1 US20060040883 A1 US 20060040883A1 US 13142505 A US13142505 A US 13142505A US 2006040883 A1 US2006040883 A1 US 2006040883A1
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wnt2
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Liang You
Biao He
Zhidong Xu
David Jablons
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University of California
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Definitions

  • This invention relates to methods of inhibiting the growth of cancer cells that overexpress Wnt2 protein.
  • the methods comprise contacting the cell with an agent that binds to Wnt2 mRNA or Wnt2 protein, interferes with Wnt2 signaling, or inhibits binding of the Wnt2 protein to other proteins, such as the Frizzled receptor.
  • Lung Cancer is the leading cause of cancer death in the United States and worldwide, with >170,000 newly diagnosed cases each year in the US and nearly a million cases worldwide (Minna et al. Cancer Cell. 1(1):49-52 (2002)). Despite aggressive approaches made in the therapy of lung cancer in the past decades, the 5-year survival rate for lung cancer remains under 15% (Minna et al. Cancer Cell. 1(1):49-52 (2002)).
  • Lung cancers are diviuded into two groups: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC).
  • NSCLC 75-80% of all cancers
  • MPM Malignant pleural mesothelioma
  • Molecular pathogenesis of lung cancer and MPM includes alterations of expression and function of multiple genes, involving dominant oncogenes and recessive tumor suppressor genes, and abnormalities in cell signaling transduction pathways. A better understanding of molecular mechanisms for lung cancer and MPM pathogenesis should improve the treatment of patients with lung cancer.
  • the Wingless-type (Wnt) family of secreted glycoproteins is a group of signaling molecules broadly involved in developmental processes and oncogenesis (Polakis, Genes Dev. 14:1837-51 (2000); Lustig et al. J. Cancer Res. Clin. Oncol. 129:199-221 (2003)).
  • Wnt proteins Nineteen human Wnt proteins have thus far been identified.
  • Transduction of Wnt signals is triggered by the binding of Wnt ligands to two distinct families of cell-surface receptors: the frizzled (Fz) receptor family and the LDL-receptor-related protein (LRP) family (Akiyama, Cytokine Growth Factor Rev. 11:273-82 (2000)).
  • Wnt signaling activates dishevelled (Dvl) proteins, which inhibit glycogen synthase kinase-3 ⁇ (GSK-3 ⁇ ) phosphorylation of ⁇ -catenin leading to its cytosolic stabilization. Stabilized ⁇ -catenin then enters the cell nucleus and associates with LEF/TCF transcription factors. ⁇ -catenin-Tcf/Lef induces transcription of important downstream target genes, many of which have been implicated in cancer.
  • Dvl dishevelled proteins
  • cytosolic ⁇ -catenin is incorporated into a complex consisting of Axin, the adenomatous polyposis coli (APC) gene product, and glycogen synthase kinase (GSK)-3 ⁇ .
  • Axin the adenomatous polyposis coli
  • GSK-3 ⁇ glycogen synthase kinase
  • Disheveled is a positive mediator of Wnt signaling positioned downstream of the frizzled receptors and upstream of ⁇ -catenin.
  • GSK-3 phosphorylates several proteins in the Wnt pathway and is instrumental in the downstream regulation of ⁇ -catenin.
  • Mutations in the gene APC are an initiating event for both sporadic and hereditary colorectal tumorigenesis. APC mutants are relevant in tumorigenesis, since the aberrant protein is an integral part of the Wnt-signaling cascade.
  • the protein product contains several functional domains acting as binding and degradation sites for ⁇ -catenin.
  • Mutations that occur in the amino-terminal segment of ⁇ -catenin are usually involved in phosphorylation-dependent, ubiquitin-mediated degradation and, thus, stabilize ⁇ -catenin.
  • stabilized cytoplasmic-catenin When stabilized cytoplasmic-catenin accumulates, it translocates to the nucleus interacting with the Tcf/Lef high-mobility group of transcription factors that modulate expression of oncogenes such as c-myc.
  • Wnt signaling pathway is also thought to be associated with tumor development and/or progression (Bienz et al., Cell 103(2):311-20 (2000); Cox et al., Genetics 155(4):1725-40 (2000); (Polakis, Genes Dev 14(15):1837-51 (2000); You t al., J Cell Biol 157(3): 429-40 (2002)).
  • c-Myc was identified as one of the transcriptional targets of the ⁇ -catenin/Tcfin colorectal cancer cells (He et al., Science 281(5382):1509-12 (1998); Miller et al., Oncogene 18(55):7860-72 (1999); You et al., J Cell Biol 157(3): 429-40 (2002)).
  • sFRPs secreted Frizzled-related proteins
  • sFRPs can either antagonize Wnt function by binding the protein and blocking access to its cell surface signaling receptor, or they can enhance Wnt activity by facilitating the presentation of ligand to the Frizzled receptors (Uthoff et al., Int J Onco l 19(4):803-10 (2001)). sFRPs seem to modulate apoptosis susceptibility, exerting an antagonistic effect on programmed cell death. To date, sFRPs have not yet been linked causatively to cancer.
  • sFRPs are reported to be hypermethylated with a high frequency in colorectal cancer cell lines and this hypermethylation is associated with a lack of basal sFRP expression (Suzuki et al., Nat Genet 31(2):141-9 (2002)).
  • Dkk-1 Another protein called Dickkopf (Dkk) is also found to interfere with Wnt signaling and diminish accumulation of cytosolic ⁇ -catenin (Moon et al., Cell 88(6):725-8 (1997); Fedi et al., J Biol Chem 274(27):19465-72 (1999)).
  • Dkk-1 antagonizes Wnt-induced signals by binding to a LDL-receptor-related protein 6 (LRP6) adjacent to the Frizzled receptor (Nusse, Nature 411(6835):255-6 (2001)).
  • LRP6 LDL-receptor-related protein 6
  • Overexpression of Dkk-1 is also found to sensitize brain tumor cells to apoptosis (Shou et al., Oncogene 21(6):878-89 (2002)).
  • Wnt proteins The effects of Wnt proteins on cell proliferation and tumor growth seem to depend on Wnt proteins interacting with their cognate cell surface receptors and subsequently inducing downstream signaling. With Wnt proteins being secreted ligands antibodies may be used to interfere with or inhibit Wnt binding to its cell surface receptor and thus affect downstream signaling.
  • Wnt proteins Several antibodies against Wnt proteins have been generated. For example, anti-Wnt-1 (G-19) (sc-6280; Santa Cruz Biotechnology, Inc.) and anti Wnt2 (H-20) (sc-5208; Santa Cruz Biotechnology, Inc.) are goat polyclonal antibodies raised against peptides mapping near the N-terminus of human Wnt-1 and Wnt2 proteins, respectively.
  • Wnt2 V-16 is a goat polyclonal antibody raised against a peptide mapping within an internal region of Wnt2 of human origin (sc-5207; Santa Cruz Biotechnology, Inc.).
  • human cell lines expressing immunoglobulins are relatively unstable compared to mouse cell lines, and the antibody producing capability of these human cell lines is transient.
  • human immunoglobulins are highly desirable, human hybridoma techniques have not yet reached the stage where human monoclonal antibodies with the required antigenic specificities can be easily obtained.
  • antibodies of non-human origin have been genetically engineered to create chimeric or humanized antibodies. Such genetic engineering results in antibodies with a reduced risk of a HAMA response compared to that expected after injection of a human patient with a mouse antibody.
  • chimeric antibodies can be formed by grafting nor-human variable regions to human constant regions (Khazaeli et al. (1991), J. Immunotherapy 15:42-52).
  • humanized antibodies are formed by grafting non-human Complementarity Determining Regions (CDRs) onto human Framework Regions (FRs) (See, Jones et al. (1986), Nature 321:522-525; and Reichman et al. (1988), Nature 332:323-327).
  • CDRs Complementarity Determining Regions
  • FRs Framework Regions
  • humanized monoclonal antibodies are formed by grafting all six (three light chain and three heavy chain) CDRs form a non-human antibody into Framework Regions (FRs) of a human antibody(e.g., see, U.S. Pat. No. 6,407,213).
  • Fv antibodies See, U.S. Pat. No. 4,642,334) or single chain Fv (SCFV) antibodies (See, U.S. Pat. No. 4,946,778) can be employed to reduce the risk of a HAMA response.
  • This invention provides a method of inhibiting the proliferation of a cell that overexpresses a Wnt2.
  • the method comprises contacting the cell with an amount of an agent that inhibits Wnt2 signaling effective to inhibit proliferation of the cell.
  • the cell is a cancer cell.
  • the cancer cell is selected from the group consisting of breast, ovarian, colorectal, gastric, lung, kidney, bladder, prostate, uterine, thyroid, pancreatic, cervical, esophageal, mesothelioma, head and neck, hepatocellular, melanoma, brain, vulval, testicular, sarcoma, intestine, skin, leukemia, and lymphoma cancer cells.
  • the agent is a siRNA.
  • the agent is an anti-Wnt2 antibody, for example, an antibody that specifically binds to the Wnt2 protein, preferably a human Wnt2 protein.
  • Antibodies of the invention can be polyclonal or monoclonal antibodies. Preferred are anti-Wnt2 monoclonal antibodies. Preferred monoclonal antibodies comprise (i) aV L CDR1 amino acid sequence as shown in SEQ ID NO:56, SEQ ID NO:104 or SEQ ID NO:125; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:60, SEQ ID NO:84, SEQ ID NO:107, SEQ ID NO:126 or SEQ ID NO:138; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63 or SEQ ID NO:87; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65 or SEQ ID NO:89; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:67, SEQ ID NO:91 or SEQ ID NO:110
  • the invention further provides anti-Wnt2 antibodies that specifically bind a polypeptide comprising an amino acid sequence corresponding to amino acid residues 49-63 of human Wnt2 (SEQ ID NO:2).
  • Another anti-Wnt2 antibody specifically binds a polypeptide comprising SSQRQLCHRPDVMR (SEQ ID NO:2).
  • This invention also provides an anti-Wnt2 antibody that competes for binding a Wnt2 protein with a second anti-Wnt2 antibody that specifically binds a polypeptide comprising an amino acid sequence corresponding to amino acid residues 49-63 of human Wnt2 (SEQ ID NO:2).
  • Another anti-Wnt2 antibody competes for binding a Wnt2 protein with a second anti-Wnt2 antibody that specifically binds a polypeptide comprising SSQRQLCHRPDVMR (SEQ ID NO:2).
  • the invention further provides a method of inducing apoptosis of a cell that overexpresses a Wnt2.
  • This method comprises contacting the cell with an amount of an agent that inhibits Wnt2 signaling effective to induce apoptosis of the cell.
  • a method of inhibiting Wnt2 signaling in a cell comprises contacting the cell that overexpresses a Wnt2 with an amount of an anti-Wnt2 antibody effective to inhibit Wnt2 signaling.
  • a method of treating a disease associated with Wnt2 signaling comprises administering to a subject in need of such treatment an amount of an agent that inhibits Wnt2 signaling effective to treat the disease.
  • the agent can be an anti-Wnt2 antibody or a siRNA.
  • the invention further provides a monoclonal anti-Wnt2 antibody that specifically binds a polypeptide comprising an amino acid sequence that corresponds to amino acid residues 49-63 of human Wnt2 (SEQ IDNO:2).
  • this monoclonal anti-Wnt2 antibody competes for binding to a Wnt2 protein with a second anti-Wnt2 antibody that specifically binds a polypeptide comprising an amino acid sequence that corresponds to amino acid residues 49-63 of human Wnt2 (SEQ IDNO:2).
  • a nucleic acid encoding an anti-Wnt2 antibody comprises (i) a nucleic acid encoding aV L CDR1 selected from the group consisting of nucleic acid sequences shown in SEQ ID NO:70, SEQ ID NO:113 and SEQ ID NO:131; (ii) a nucleic acid encoding aV L CDR2 selected from the group consisting of nucleic acid sequences shown in SEQ ID NO:72 and SEQ ID NO:115; (iii) a nucleic acid encoding a V L CDR3 selected from the group of nucleic acid sequences shown in SEQ ID NO:94, SEQ ID NO:117, SEQ ID NO:134 and SEQ ID NO:143; (iv) a nucleic acid encoding aV H CDR1 selected from the group of nucleic acid sequences shown in SEQ ID NO:77 and S
  • compositions comprising an anti-Wnt2 antibody or a siRNA and a pharmaceutically acceptable excipient, carrier and/or diluent.
  • FIG. 1 Anti-Wnt2 monoclonal antibody induces apoptosis in cancer cells.
  • This panel shows 0.5% Crystal Violet staining of NHBE cells and four human NSCLC cell lines (A549, H460, H1703, and H1299) after the anti-Wnt2 antibody treatment.
  • the panel shows these examples of apoptosis analysis by flow cytometry. From top to bottom, A549, H460, and H1703 NSCLC cells were treated with control antibody and anti Wnt2 monoclonal antibody, respectively.
  • the panel shows percentage of dead A549 cells after about 72 h treatment with monoclonal antibody alone and with monoclonal antibody blocked by preincubation with blocking peptide. Controls are blocking peptide alone and control mAb. After incubation, cells were collected for flow cytometry analysis. Results are the means ⁇ s.d. (error bars).
  • FIG. 3 Wnt2 siRNA induces apoptosis and blocks Wnt signal transduction in NSCLC cell line A549.
  • FIG. 4 In vivo study of anti Wnt2 monoclonal antibody on lung metastasis.
  • FIG. 5 Synergy with chemotherapy. A synergistic effect in A549 cells is observed when low dose chemotherapy (docetaxel) is combined with low dose of monoclonal anti Wnt2 antibody.
  • FIG. 6 The anti-Wnt2 monoclonal antibody induces apoptosis in melanoma cell lines and primary cultures.
  • This panel shows 0.5% Crystal Violet staining of normal human epithelial keratinocytes (NHEK) and four human melanoma cell lines (LOX, FEMX, FEM, and SK-Mel-2) after the anti-Wnt2 antibody treatment.
  • LOX, FEMX, FEM, and SK-Mel-2 human melanoma cell lines
  • the panel shows these examples of apoptosis analysis by flow cytometry. FEMX and LOX cancer cells were treated with control antibody and anti Wnt2 monoclonal antibody, respectively.
  • FL1-H represents annexin V-FITC staining.
  • FIG. 7 Wnt2 siRNA induces apoptosis and blocks Wnt signal transduction in FEMX and LOX cancer cell lines.
  • c-Myc and fibronectin genes were down-regulated after Wnt2 siRNA treatment in LOX cells. Total RNA was used for hybridization.
  • FIG. 8 Anti-Wnt2 monoclonal antibody suppresses tumor growth in vivo.
  • FIG. 9 Wnt-related gene expression profile in mesothelioma tissues.
  • A RNA was extracted from 16 matched malignant mesothelioma and adjacent normal pleura. After extraction, RNA was subjected to a reverse transcriptase reaction and cDNA probes were labeled with Biotin-16-dUTP, and hybridized with the Wnt specific arrays. Detection was done using a chemiluminescent reaction and the membranes were exposed to X-ray film. Here are shown two matched samples as an example. Wnt2 is surrounded by a black circle.
  • B Data were then matched against the gene list of the GEArray Q series human Wnt signaling pathway array provided by the manufacturer. Upregulated genes in the malignant tissue compared with the normal tissue are shown in gray (light grey for Wnt2).
  • C Upregulated and downregulated genes in all studied samples (8 pairs) are detailed.
  • FIG. 10 Wnt2 expression in normal and mesothelioma cell lines and in tissues.
  • A Western Blot analysis of Wnt2 expression in normal mesothelial cell line (LP9) and in several malignant mesothelioma cell lines.
  • B Western Blot analysis of Wnt2 expression in tissues. Whole cell extracts were prepared from freshly resected tumor (T) and autologous matched normal pleura (N). Actin was used as a control.
  • FIG. 11 Anti-Wnt2 monoclonal antibody induces apoptosis in mesothelioma cancer cell lines.
  • A Staining with 0.5% Crystal Violet of four malignant pleural mesothelioma cell lines either untreated or treated with a control antibody (control Ab) or a specific monoclonal anti-Wnt2 antibody at a concentration of 10 ⁇ g/ml (Wnt2 mAb).
  • control Ab control antibody
  • Wnt2 mAb a specific monoclonal anti-Wnt2 antibody at a concentration of 10 ⁇ g/ml
  • FIG. 12 Wnt2 siRNA induces apoptosis and blocks Wnt signal transduction in mesothelioma cell lines.
  • A 0.5% Crystal Violet staining of three malignant pleural mesothelioma cell lines realized 35 days after transfection with lipofectamine alone (untreated), with a non-silencing siRNA (control siRNA) or 100 nM of a specific Wnt2 siRNA (Wnt2 siRNA).
  • B Annexin V analysis of apoptosis induced by Wnt2 siRNA.
  • Mesothelioma cells were transfected as described in A.
  • FIG. 13 Effects of Wnt2 antibody and Alimta® on cell proliferation.
  • MS1 mesothelioma cell line was plated in 96-well plates and treated with increasing concentration of Wnt2 antibody (dotted line), Alimta® (solid line) or both (dashed line).
  • Cell proliferation was assessed 3 days latter by measuring the metabolic activity of cellular enzyme (here the tetrazolium conversion). Results are the means ⁇ SD (error bars).
  • FIG. 14 Amino acid and nucleic acid sequences of Complementarity Determining Regions (CDR) and Framework Regions (FR) of anti-Wnt2 monoclonal antibody 17F7.G7 (subclone A). Sequences for the light chain kappa and the heavy chain IgG 1 are shown. The numbering refers to the position of amino acid residues based on alignment with a mouse monoclonal antibody (IMGT, Immunogen Genetics). FR regions are indicated by interrupted lines, CDR regions by solid lines.
  • FIG. 15 Amino acid and nucleic acid sequences of Complementarity Determining Regions (CDR) and Framework Regions (FR) of anti-Wnt2 monoclonal antibody 8B11.D2.
  • CDR Complementarity Determining Regions
  • FR Framework Regions
  • FR regions are indicated by interrupted lines, CDR regions by solid lines.
  • FIG. 16 Amino acid and nucleic acid sequences of Complementarity Determining Regions (CDR) and Framework Regions (FR) of anti-Wnt2 monoclonal antibody 17F7.E5.
  • CDR Complementarity Determining Regions
  • FR Framework Regions
  • FIG. 17 Amino acid and nucleic acid sequences of Complementarity Determining Regions (CDR) and Framework Regions (FR) of anti-Wnt2 monoclonal antibody 8B11.H6.
  • A Sequences for the light chain kappa (Chain-1).
  • B Sequences for the light chain kappa (Chain-2).
  • C Sequences for the heavy chain IgG 1 . Numbering of amino acid residues is as described in FIG. 14 .
  • FR regions are indicated by interrupted lines, CDR regions by solid lines.
  • FIG. 18 Amino acid and nucleic acid sequences of Complementarity Determining Regions (CDR) and Framework Regions (FR) of anti-Wnt2 monoclonal antibody 17F7.G7 (subclone B).
  • CDR Complementarity Determining Regions
  • FR Framework Regions
  • C Sequences for the heavy chain IgG 1 . Numbering of amino acid residues is as described in FIG. 14 .
  • FR regions are indicated by interrupted lines, CDR regions by solid lines.
  • Wnt protein or “Wnt ligand” refer to a family of mammalian proteins related to the Drosophila segment polarity gene, wingless. In humans, the Wnt family of genes typically encode 38 to 43 kDa cysteine rich glycoproteins having hydrophobic signal sequence, and a conserved asparagine-linked oligosaccharide consensus sequence (Shimizu et al., Cell Growth Differ 8(12):1349-58 (1997)). The Wnt family contains at least 19 mammalian members.
  • Exemplary Wnt proteins include Wnt1, Wnt2, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, WNT10A, Wnt10B, Wnt11, Wnt12, Wnt13, Wnt14, Wnt15, and Wnt16.
  • a preferred Wnt protein of the invention is Wnt2, preferably a human Wnt2 protein.
  • Frizzled protein or “frizzled receptor” refer to a family of mammalian proteins related to the Drosophila frizzled genes, which play a role in the development of tissue polarity.
  • the Frizzled family comprises at least 10 mammalian genes.
  • Exemplary human Frizzled receptors include Frizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5, Frizzled6, Frizzled7, Frizzled8, Frizzled9 and Frizzled10.
  • Frizzled1 Frizzled2, Frizzled3, Frizzled4, Frizzled5, Frizzled6, Frizzled7, Frizzled8, Frizzled9 and Frizzled10.
  • the mammalian homologues of the Drosophila frizzled protein share a number of common structural motifs.
  • the N-terminus located at the extracellular membrane surface is followed by a signal sequence, a domain of 120 amino acids with an invariant pattern of 10 cysteine residues, and a highly divergent region of 40-100 largely variable hydrophilic amino acids.
  • Putative hydrophobic segments form seven membrane-spanning helices linked by hydrophilic loops, ending with the C terminus located at the intracellular face of the membrane.
  • the cysteine-rich domains (CRDs) and the transmembrane segments are strongly conserved, suggesting a working model in which an extracellular CRD is tethered by a variable linker region to a bundle of seven membrane-spanning helices.
  • Frizzled protein receptors are, therefore, involved in a dynamic model of transmembrane signal transduction analogous to G-protein-coupled receptors with amino-terminal ligand binding domains.
  • Frizzled1, Frizzled2, and Frizzled7 are involved in lung and colorectal cancers, (Sagara et al., Biochem Biophys Res Commun 252(1):117-22 (1998)); Frizzled3 in human cancer cells including lung, cervical and colorectal cancers, (Kirikoshi et al., Int J Oncol 19(4):767-71 (2001)); Frizzled7 in gastric cancer (Kirikoshi et al., Int J Oncol 19(4):767-71 (2001)); Frizzled10 in gastric and colorectal cancer, Kirikoshi et al., Int J Oncol 19(4):767-71 (2001); Terasaki et al., Int J Mol Med 9(2):107-12 (2002).
  • Dvl refers to a member of a family of Dishevelled proteins, the full-length sequences of which typically possess three conserved domains, a DIX domain, present in the Wnt antagonizing protein Axin; a PDZ domain involved in protein-protein interactions, and a DEP domain found in proteins that regulate Rho GTPases.
  • Dvl proteins include, for example, Dvl- 1, Dvl-2, and Dvl-3.
  • Nucleic acid and protein Dvl sequence are known from a variety of species, including mouse and human. Exemplary human Dvl-1, Dvl-2, and Dvl-3 protein sequences are available under reference sequences NP — 004412, NP — 004413, and NM — 004414, respectively.
  • “Inhibitors” of Wnt signaling and in particular Wnt2 signaling refers to compounds or agents that, e.g., bind to Wnt or Frizzled proteins, or partially or totally block Wnt signaling as measured in known assays for Wnt signaling (e.g., measurement of ⁇ -catenin levels, or oncogene expression controlled by Tcf and Lef transcription factors). Inhibitors, include modified versions of Wnt or Frizzled proteins, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, small chemical molecules, and the like. Assays for detecting inhibitors of the invention are described in more detail below.
  • cell that overexpresses Wnt2 protein refers to a cell or cancer cell in which expression of a Wnt2 protein or Wnt2 mRNA is at least about 2 times, usually at least about 5 times the level of expression in a normal cell from the same tissue.
  • Methods for determining the level of expression of a particular gene are well known in the art. Such methods include RT-PCR, use of antibodies against the gene products, and the like.
  • antibody includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies).
  • the term “antibody” also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab′, F(ab′) 2 , Fab, Fv and rIgG. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.).
  • antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.
  • An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen.
  • an immunoglobulin typically has a heavy and light chain.
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains” ).
  • Light and heavy chain variable regions contain four “framework” regions interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.”
  • CDRs complementarity-determining regions
  • the extent of the framework regions and CDRs have been defined.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • a V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • V H or a “VH” refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv (disulfide-stabilized Fv) or Fab.
  • V L or a “V L ” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
  • single chain Fv or “scFv” refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.
  • a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
  • a “chimeric antibody” is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a “humanized antibody” is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
  • Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • Fully human antibody refers to an immunoglobulin comprising human variable regions in addition to human framework and constant regions.
  • 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., McCafferty et al., 1990, Nature 348:552-554; Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol. Biol.
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Pat. Nos.
  • Epitope or “antigenic determinant” refers to a site on an antigen to which an antibody binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
  • Biological sample as used herein is a sample of biological tissue or fluid that contains nucleic acids or polypeptides, e.g., of a Wnt protein, polynucleotide or transcript. Such samples include, but are not limited to, tissue isolated from primates, e.g., humans, or rodents, e.g., mice, and rats. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues.
  • a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
  • a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
  • “Providing a biological sample” means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, preferably a human, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful.
  • the “level of Wnt2 mRNA” in a biological sample refers to the amount of mRNA transcribed from a Wnt2 gene that is present in a cell or a biological sample.
  • the mRNA generally encodes a functional Wnt2 protein, although mutations may be present that alter or eliminate the function of the encoded protein.
  • a “level of Wnt2 mRNA” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • the “level of Wnt2 protein or polypeptide” in a biological sample refers to the amount of polypeptide translated from Wnt2 mRNA that is present in a cell or biological sample.
  • the polypeptide may or may not have Wnt2 protein function.
  • a “level of Wnt2 protein” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., Altschul et al., Nucl.
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man-made variants.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of one of the number of contiguous positions selected from the group consisting typically of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • Log values may be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, etc.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene.
  • purified in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • “Purify” or “purification” in other embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.
  • 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 an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor & Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
  • Primary structure refers to the amino acid sequence of a particular peptide.
  • “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that often form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long.
  • Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ -helices.
  • Tetiary structure refers to the complete three dimensional structure of a polypeptide monomer.
  • Quaternary structure refers to the three dimensional structure formed, usually by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • the radioisotope may be, for example, 3 H, 14 C, 32 p, 35 S, or 125 I.
  • the radioisotopes are used as toxic moieties, as described below.
  • the labels may be incorporated into the nucleic acids, proteins and antibodies at any position. Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).
  • radiolabeled peptides or radiolabeled antibody compositions may extended by the addition of substances that stablize the radiolabeled peptide or antibody and protect it from degradation. Any substance or combination of substances that stablize the radiolabeled peptide or antibody may be used including those substances disclosed in U.S. Pat. No. 5,961,955.
  • effector or “effector moiety” or “effector component” is a molecule that is bound (or linked, or conjugated), either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, to an antibody.
  • the “effector” can be a variety of molecules including, e.g., detection moieties including radioactive compounds, fluorescent compounds, an enzyme or substrate, tags such as epitope tags, a toxin; activatable moieties, a chemotherapeutic agent; a lipase; an antibiotic; or a radioisotope emitting “hard” e.g., beta radiation.
  • recombinant when used with reference to, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • nucleic acid By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved.
  • an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the purposes of this invention.
  • a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
  • a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • an antibody or antigen such as a protein, preferably a Wnt2 protein or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide or antibody, refers to a binding reaction that is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologics.
  • the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background.
  • antibodies raised to a particular protein polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof, can be selected to obtain only those antibodies that are specifically immunoreactive with Wnt2 proteins and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • Tumor cell refers to precancerous, cancerous, and normal cells in a tumor.
  • “Cancer cell,““transformed” cell or “transformation” in tissue culture” refers to spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material.
  • transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.
  • transformation is typically associated with overexpression of Wnt and/or Frizzled proteins. Transformation is associated with other phenotypic changes, such as immortalization of cells, aberrant growth control, nonmorphological changes, and/or malignancy (see, Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).
  • small interfering RNA or “siRNA” is meant an isolated RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that has been shown to function as a key intermediate in triggering sequence-specific RNA degradation. A range of 19-25 nucleotides is the most preferred size for siRNAs.
  • siRNAs can also include short hairpin RNAs (shRNA) in which both strands of an siRNA duplex are included within a single RNA molecule. Double-stranded siRNAs generally consist of a sense and anti-sense strand.
  • siRNAs generally consist of only the antisense strand that is complementary to the target gene or mRNA.
  • siRNA includes any form of RNA, preferably dsRNA (proteolytically cleaved products of larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides.
  • the role of Wnt-Fz signaling pathway in oncogenesis has been described to some extent in WO 04/032838.
  • the present invention provides inhibitors of Wnt2 signaling pathway that can induce apoptosis in a number of cancers overexpressing Wnt2.
  • the invention is useful for treatment of a disease associated with Wnt2 signaling, in particular a cancer in which Wnt2 signaling affects cancer cell growth or survival.
  • the invention is particularly useful for treating cancers such as breast cancer, ovarian cancer, colorectal cancer, gastric cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, uterine cancer, thyroid cancer, pancreatic cancer, cervical cancer, esophageal cancer, mesothelioma, head and neck cancer, hepatocellular carcinoma, melanoma, brain cancer, vulval cancer, testicular cancer, sarcoma, intestine cancer, skin cancer, leukemia, and lymphoma cancer.
  • cancers such as breast cancer, ovarian cancer, colorectal cancer, gastric cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, uterine cancer, thyroid cancer, pancreatic cancer, cervical cancer, esophageal cancer, mesothelioma, head and neck cancer, hepatocellular carcinoma, melanoma, brain cancer, vulval cancer, testicular cancer, sarcoma, intestine cancer, skin cancer, leukemia, and lymphoma cancer
  • Blocking Wnt2 signaling is shown here to lead to down-regulation of downstream components of the Wnt-Fz pathway, in particular, Dishevelled (Dvl) and ⁇ -catenin.
  • This invention also shows that antibody-induced apoptosis occurs through activation of JNK, releasing Smac/Diablo and cytochrome C from mitochondria to the cytosol. Cytochrome C inactivates survivin, an inhibitor of apoptosis, that leads to the activation of caspases.
  • the invention further provides anti-Wnt2 monoclonal antibodies that suppress growth of tumors in vivo.
  • Antibodies that may be used in the methods, pharmaceutical compositions and kits of the present invention may be polyclonal anti-Wnt2 antibodies or monoclonal anti-Wnt2 antibodies.
  • the antibodies are monoclonal anti-Wnt2 antibodies.
  • Monoclonal antibodies of the invention may be prepared in a variety of ways. A preferred method uses hybridoma methods, such as those described by Köhler & Milstein, Nature 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • a Wnt2 monoclonal antibody comprises a (i) aV L CDR1 amino acid sequence as shown in SEQ ID NO:56, SEQ ID NO:104 or SEQ ID NO:125; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:60, SEQ ID NO:84, SEQ ID NO:107, SEQ ID NO:126 or SEQ ID NO:138; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63 or SEQ ID NO:87; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65 or SEQ ID NO:89; or (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:67, SEQ ID NO:91 or SEQ ID
  • a monoclonal Wnt2 antibody comprises a (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:56; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; and (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:84.
  • 17F7.G7 subclone B, Chain 2
  • FIG. 17 As further exemplified by 17F7.G7 (subclone B, Chain 2, FIG.
  • a monoclonal Wnt2 antibody comprises (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:104; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; and (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:138.
  • a V L CDR1 amino acid sequence as shown in SEQ ID NO:104
  • a V L CDR2 amino acid sequence as shown in SEQ ID NO:58
  • Viii a V L CDR3 amino acid sequence as shown in SEQ ID NO:138.
  • a monoclonal Wnt2 antibody may also comprises (i) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63; (ii) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65; and (iii) a V H CDR3 amino acid sequence as shown in SEQ ID NO:110.
  • a V H CDR1 amino acid sequence as shown in SEQ ID NO:63
  • a V H CDR2 amino acid sequence as shown in SEQ ID NO:65
  • Viii a V H CDR3 amino acid sequence as shown in SEQ ID NO:110.
  • the anti-Wnt2 monoclonal antibody may comprise (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:56; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:84; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:110.
  • Another anti-Wnt2 monoclonal antibody comprises (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:104; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:138; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:110.
  • an anti-Wnt2 monoclonal antibody comprises (i) aV L CDR1 amino acid sequence as shown in SEQ ID NO:104; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:107; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:110.
  • Another preferred anti-Wnt2 monoclonal antibody exemplified by anti-Wnt2 monoclonal antibody 8B11.D2 ( FIG. 15 ) comprises (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:56; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; and (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:84.
  • anti-Wnt2 monoclonal antibodies 8B11.D2 ( FIG. 15 ) and 8B11.H6 Choin 1, FIG.
  • an anti-Wnt2 monoclonal antibody may also comprise (i) a V H CDR1 amino acid sequence as shown in SEQ ID NO:87; (ii) a V H CDR2 amino acid sequence as shown in SEQ ID NO:89; and (iii) a V H CDR3 amino acid sequence as shown in SEQ ID NO:91.
  • an anti-Wnt2 monoclonal antibody comprises (i) aV L CDR1 amino acid sequence as shown in SEQ ID NO:56; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:84; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:87; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:89; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:91.
  • an anti-Wnt2 monoclonal antibody comprises (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:56; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:84; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:87; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:89; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:91.
  • Another preferred anti-Wnt2 monoclonal antibody exemplified by 8B11.H6 (Chain 2, FIG. 17 ), comprises (i) aV L CDR1 amino acid sequence as shown in SEQ ID NO:125; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; and (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:126.
  • Another Wnt-2 monoclonal antibody comprises (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:125; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; and (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:126; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:87; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:89; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:91.
  • anti-Wnt2 antibodies may be generated using a Wnt2 peptide comprising any of the amino acid sequences of SEQ ID NOS:1-15 and 30-53.
  • a preferred anti-Wnt2 antibody is an antibody that specifically binds to a polypeptide comprising an amino acid sequence corresponding to amino acid residues 49-63 of human Wnt2 (SEQ ID NO:2).
  • the phrase “corresponding to amino acid residues 49-63 of human Wnt2 (SEQ ID NO:2)” refers to an amino acid sequence of a non-human Wnt2 protein or another Wnt protein, wherein the respective amino acid sequence corresponds to amino acid residues 49-63 of human Wnt2 (SEQ ID NO:2).
  • amino acid sequences may have one or more amino acid differences when compared to amino acid residues 49-63 of human Wnt2 (SEQ ID NO:2).
  • an anti-Wnt2 antibody specifically binds a polypeptide comprising SSQRQLCHRPDVMR (SEQ ID NO:2), and in particular binds to the SSQRQLCHRPDVMR (SEQ ID NO:2) epitope of the polypeptide.
  • Anti-Wnt2 antibodies of the present invention can be prepared in a variety of ways.
  • anti-Wnt2 antibodies are produced recombinantly.
  • the anti-Wnt2 antibody is encoded by a nucleic acid that is inserted into an expression vector and expressed in a suitable host as known in the art.
  • a nucleic acid encoding an anti-Wnt2 monoclonal antibody comprises a nucleic acid comprising (i) a nucleic acid encoding a V L CDR1 as shown in SEQ ID NO:70; (ii) a nucleic acid encoding a V L CDR2 as shown in SEQ ID NO:72; and (iii) a nucleic acid encoding a V L CDR3 as shown in SEQ ID NO:94.
  • a nucleic acid encoding an anti-Wnt2 monoclonal antibody may also comprise (i) a nucleic acid encoding a V H CDR1 as shown in SEQ ID NO:97; (ii) a nucleic acid encoding a V H CDR2 as shown in SEQ ID NO:99; and (iii) a nucleic acid encoding a V H CDR3 as shown in SEQ ID NO:101.
  • a nucleic acid encoding an anti-Wnt2 monoclonal antibody comprises a nucleic acid comprising (i) a nucleic acid encoding a V L CDR1 as shown in SEQ ID NO:70; (ii) a nucleic acid encoding a V L CDR2 as shown in SEQ ID NO:72; and (iii) a nucleic acid encoding a V L CDR3 as shown in SEQ ID NO:94; (iv) a nucleic acid encoding a V H CDR1 as shown in SEQ ID NO:97; (v) a nucleic acid encoding a V H CDR2 as shown in SEQ ID NO:99; and (vi) a nucleic acid encoding a V H CDR3 as shown in SEQ ID NO:101.
  • nucleic acid encoding an anti-Wnt2 monoclonal antibody comprises a nucleic acid comprising (i) a nucleic acid encoding a V L CDR1 as shown in SEQ ID NO:131; (ii) a nucleic acid encoding a V L CDR2 as shown in SEQ ID NO:115; and (iii) a nucleic acid encoding a V L CDR3 as shown in SEQ ID NO:134.
  • a nucleic acid encoding an anti-Wnt2 monoclonal antibody comprises a nucleic acid comprising (i) a nucleic acid encoding a V L CDR1 as shown in SEQ ID NO:70; (ii) a nucleic acid encoding a V L CDR2 as shown in SEQ ID NO:72; and (iii) a nucleic acid encoding a V L CDR3 as shown in SEQ ID NO:94.
  • a nucleic acid encoding an anti-Wnt2 monoclonal antibody may also comprise (i) a nucleic acid encoding a V H CDR1 as shown in SEQ ID NO:77; (ii) a nucleic acid encoding a V H CDR2 as shown in SEQ ID NO:79; and (iii) a nucleic acid encoding a V H CDR3 as shown in SEQ ID NO:120.
  • a nucleic acid encoding an anti-Wnt2 monoclonal antibody comprises a nucleic acid comprising (i) a nucleic acid encoding a V L CDR1 as shown in SEQ ID NO:70; (ii) a nucleic acid encoding a V L CDR2 as shown in SEQ ID NO:72; (iii) a nucleic acid encoding a V L CDR3 as shown in SEQ ID NO:94; (iv) a nucleic acid encoding a V H CDR1 as shown in SEQ ID NO:77; (v) a nucleic acid encoding a V H CDR2 as shown in SEQ ID NO:79; and (vi) a nucleic acid encoding a V H CDR3 as shown in SEQ ID NO:120.
  • Another preferred nucleic acid encoding an anti-Wnt2 monoclonal antibody comprises a nucleic acid comprising (i) a nucleic acid encoding a V L CDR1 as shown in SEQ ID NO:113; (ii) a nucleic acid encoding a V L CDR2 as shown in SEQ ID NO:115; and (iii) a nucleic acid encoding a V L CDR3 as shown in SEQ ID NO:143; (iv) a nucleic acid encoding a V H CDR1 as shown in SEQ ID NO:77; (v) a nucleic acid encoding a V H CDR2 as shown in SEQ ID NO:79; and (vi) a nucleic acid encoding a V H CDR3 as shown in SEQ ID NO:120.
  • a nucleic acid encoding an anti-Wnt2 monoclonal antibody comprises a nucleic acid comprising (i) a nucleic acid encoding a V L CDR1 as shown in SEQ ID NO:113; (ii) a nucleic acid encoding a V L CDR2 as shown in SEQ ID NO:115; and (iii) a nucleic acid encoding a V L CDR3 as shown in SEQ ID NO:117.
  • anti Wnt2 monoclonal antibodies can be produced recombinantly.
  • a chimeric or humanized anti-Wnt2 monoclonal antibody is produced recombinantly.
  • Recombinant DNA technology may be employed wherein a nucleotide sequence that encodes an anti-Wnt2 monoclonal antibody or a fragment thereof, such as one or more CDR sequences, is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • Vector refers to any type of genetic construct containing a nucleic acid capable of being transcribed in a cell. Vectors used for the amplification of nucleotide sequences (both coding and non-coding) are also encompassed by the definition. In addition to the coding sequence, vectors will generally include restriction enzyme cleavage sites and the other initial, terminal and intermediate DNA sequences that are usually employed in vectors to facilitate their construction and use. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • any modification within a DNA or RNA sequence can be made simply by substituting the appropriate bases for those encoding the desired amino acid sequence.
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the immunostimulating peptide or protein.
  • a number of such vectors and suitable host systems are commercially available.
  • the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences as known to the skilled artisan.
  • a chimeric or humanized anti-Wnt2 antibody comprises (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:56, SEQ ID NO:104 or SEQ ID NO:125; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:60, SEQ ID NO:84, SEQ ID NO:107, SEQ ID NO:126 or SEQ ID NO:138; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63, or SEQ ID NO:87; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65 or SEQ ID NO:89; or (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:67, SEQ ID NO:91 or SEQ ID NO:110.
  • a chimeric or humanized anti-Wnt2 antibody comprises (i) a V L CDR1 amino acid sequence as shown in SEQ ID NO:56, SEQ ID NO:104 or SEQ ID NO:125; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:60, SEQ ID NO:84, SEQ ID NO:107, SEQ ID NO:126 or SEQ ID NO:138; (iv) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63, or SEQ ID NO:87; (v) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65 or SEQ ID NO:89; and (vi) a V H CDR3 amino acid sequence as shown in SEQ ID NO:67, SEQ ID NO:91 or SEQ ID NO:110.
  • chimeric or humanized anti-Wnt2 antibody comprise combinations of V L CDRs and V H CDRs as described herein and as exemplified by the anti-Wnt2 monoclonal antibodies 17F7.G7 (subclone B, Chains 1 and 2, FIG. 18 ), 8B11.H6 (Chains 1 and 2, FIG. 17 ), 17F7.E5 ( FIG. 16 ), and 8B11.D2 ( FIG. 15 ).
  • Human antibodies can be produced using various techniques known in the art, including phage display libraries (Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boemer et al., J. Immunol. 147(1):86-95 (1991)).
  • the peptide linker will have no specific biological activity other than to join the regions or to preserve some minimum distance or other spatial relationship between the V H and V L .
  • the constituent amino acids of the peptide linker may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
  • the V L region of the scFv comprises (i) aV L CDR1 amino acid sequence as shown in SEQ ID NO:56, SEQ ID NO:104 or SEQ ID NO:125; (ii) a V L CDR2 amino acid sequence as shown in SEQ ID NO:58; (iii) a V L CDR3 amino acid sequence as shown in SEQ ID NO:60, SEQ ID NO:84, SEQ ID NO:107, SEQ ID NO:126 or SEQ ID NO:138.
  • the V H region of the scFv may comprise (i) a V H CDR1 amino acid sequence as shown in SEQ ID NO:63 or SEQ ID NO:87; (ii) a V H CDR2 amino acid sequence as shown in SEQ ID NO:65 or SEQ ID NO:89; or (iii) a V H CDR3 amino acid sequence as shown in SEQ ID NO:67, SEQ ID NO:91 or SEQ ID NO:110.
  • the antibodies are bispecific antibodies.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen.
  • one of the binding specificities is for the Wnt2 protein, the other one is for another cancer antigen.
  • tetramer-type technology may create multivalent reagents.
  • Binding affinity for a target antigen is typically measured or determined by standard antibody-antigen assays, such as Biacore competitive assays, saturation assays, or immunoassays such as ELISA or RIA.
  • the binding interactions between antigen and antibody include reversible noncovalent associations such as electrostatic attraction, Van der Waals forces and hydrogen bonds.
  • the antibodies of the invention specifically bind to Wnt2 proteins.
  • “specifically bind” herein is meant that the antibodies bind to the Wnt2 protein with a K D of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • This protein will be bound to the antibody through a specific antibody:epitope interaction.
  • a second antibody which has been covalently linked to a detectable moeity (e.g., HRP, with the labeled antibody being defined as the detection antibody) is added to the ELISA. If this antibody recognizes the same epitope as the capture antibody it will be unable to bind to the target protein as that particular epitope will no longer be available for binding. If however this second antibody recognizes a different epitope on the target protein it will be able to bind and this binding can be detected by quantifying the level of activity (and hence antibody bound) using a relevant substrate.
  • a detectable moeity e.g., HRP, with the labeled antibody being defined as the detection antibody
  • the present invention also provides diagnostic assays for detecting Wnt2.
  • activity of the Wnt2 gene is determined by a measure of gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity.
  • mRNA or cDNA genes that are known to those of skill in the art.
  • one method for evaluating the presence, absence, or quantity of mRNA involves a Northern blot transfer.
  • a transcript e.g., mRNA
  • amplification e.g. PCR
  • transcript level is assessed by using reverse transcription PCR (RT-PCR). Primer pairs useful in such methods are disclosed in SEQ ID NOS:16-29.
  • the “activity” of the Wnt2 gene can also be detected and/or quantified by detecting or quantifying the expressed polypeptide.
  • the polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • hyperdiffusion chromatography and the like.
  • the isolated proteins can also be sequenced according to standard techniques to identify polymorphisms.
  • the antibodies of the invention can also be used to detect the Wnt2 protein, or cells expressing them, using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
  • the present invention provides methods of detecting cells that overexpress Wnt2, in particular cancer cells.
  • Wnt2 expression is analyzed in a biological sample.
  • a biopsy is performed on the subject and the collected tissue is tested in vitro.
  • the tissue or cells from the tissue is then contacted, with an anti-Wnt2 antibody of the invention.
  • An immune complexe which results indicates the presence of a Wnt2 protein in the biopsied sample.
  • the antibody can be radiolabeled or coupled to an effector molecule which is a detectable label, such as a radiolabel.
  • the cell or cancer cell overexpressing Wnt2 is detected in vivo using, for example, typical imaging systems. Then, the localization of the label is determined by any of the known methods for detecting the label.
  • a conventional method for visualizing diagnostic imaging can be used. For example, paramagnetic isotopes can be used for MRI. Internalization of the antibody may be important to extend the life within the organism beyond that provided by extracellular binding, which will be susceptible to clearance by the extracellular enzymatic environment coupled with circulatory clearance.
  • the methods described above can also be used in prognostic assays or to predict drug response, that is as a pharmacogenomic marker.
  • the methods can be used to predict a response to therapeutic regimens described herein.
  • such methods can be used to predict a response to therapeutic methods using the anti-Wnt2 antibodies of the invention.
  • Wnt2 signaling such as the anti-Wnt2 antibodies and siRNAs of the invention may find use in a variety of ways.
  • a method of inhibiting proliferation of a cell that overexpresses a Wnt2 is provided.
  • the Wnt2 that is overexpressed can be either a Wnt2 protein or a Wnt2 mRNA.
  • This method comprises the step of contacting the cell with an amount of an agent that inhibits Wnt2 signaling effective to inhibit proliferation of the cell.
  • “Proliferation” refers to the growth of a cell, the reproduction or multiplication of a cell or morbid cysts.
  • this method is practiced in vitro. As further described herein, the methods of the present invention can also be practiced in vivo.
  • Wnt2 signaling such as the anti-Wnt2 antibodies and siRNAs of the invention may find use in a variety of ways.
  • a method of inducing apoptosis of a cell that overexpresses a Wnt2 is provided. This method comprises the step of contacting the cell with an amount of an agent that inhibits Wnt2 signaling effective to induce apoptosis of the cell.
  • Agents for use in this method such as anti-Wnt2 antibodies or siRNAs are disclosed herein.
  • Agents of the present invention such as the anti-Wnt2 antibodies and siRNAs of the invention may find use in a variety of ways.
  • a method of inhibiting Wnt2 signaling in a cell comprises the step of contacting a cell that overexpresses a Wnt2 with an amount of an agent effective to inhibit Wnt2 signaling.
  • Agents for use in this method such as anti-Wnt2 antibodies or siRNAs are disclosed herein.
  • the disease is a cancer.
  • Agents of the present invention are useful for treating a cancer selected from the group consisting of breast, ovarian, colorectal, gastric, lung, kidney, bladder, prostate, uterine, thyroid, pancreatic, cervical, esophageal, mesothelioma, head and neck, hepatocellular, melanoma, brain, vulval, testicular, sarcoma, intestine, skin, leukemia, and lymphoma cancer.
  • a preferred cancer is breast cancer, melanoma, lung cancer, mesothelioma, thyroid cancer, colon cancer or liver cancer.
  • the terms “treat”, “treating”, and “treatment” include: (1) preventing a disease, such as cancer, i.e. causing the clinical symptoms of the disease not to develop in a subject that may be predisposed to the disease but does not yet experience any symptoms of the disease; (2) inhibiting the disease, i.e. arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e. causing regression of the disease or its clinical symptoms.
  • the subject in need of such treatment is a mammal, more preferable a human.
  • This invention also provides a method of treating a cancer that overexpresses Wnt2.
  • This method comprises the step of administering to a subject in need of such treatment an amount of an agent effective to treat the cancer.
  • Agents for use in this method such as anti-Wnt2 antibodies or siRNAs are disclosed herein.
  • detecting the level of Wnt2 expression is carried out by detecting the level of Wnt2 mRNA. In another embodiment of the invention, detecting the level of Wnt2 expression is carried out by detecting the level of Wnt2 protein.
  • Agents for use in this method such as anti-Wnt2 antibodies or siRNAs are disclosed herein.
  • Detection of the level of Wnt2 expression may be determined for a variety of reasons. Detecting the level of Wnt2 expression may be (i) part of screening, diagnosis or prognosis of cancer in the subject; (ii) part of determining susceptibility of the subject to cancer; (iii) part of determining the stage or severity of a cancer in the subject; (iv) part of identifying a risk for the subject of developing a cancer; or (v) part of monitoring the effect of an anti-cancer drug or therapy administered to the subject diagnosed with cancer.
  • the anti-cancer drug or therapy administered to the subject may comprise an anti-Wnt2 antibody or a siRNA of this invention.
  • a method for identifying in a subject the stage or severity of a cancer is provided.
  • Wnt2 expression is overexpressed in various cancer cells (e.g., Tables 3-5).
  • anti Wnt2 antibodies and siRNA induce in a dose-dependent manner apoptosis in those cells.
  • amounts of Wnt2 are characteristic of various cancer risk states, e.g., high, medium or low.
  • the stage or severity of a cancer may be determined by measuring Wnt2 and then either submitting them to a classification algorithm or comparing them with a reference amount and/or pattern of Wnt2 that is associated with a particular stage or severity of the cancer.
  • a cancer in a subject is determined as part of screening, diagnosis or prognosis of the ancer in the subject.
  • Wnt2 levels are determined in a biological sample from a subject to be screened for cancer.
  • Wnt2 compositions are useful for treatment of cancer wherein Wnt2 expression is overexpressed.
  • other drugs for example, a composition comprising an inhibitor of Wnt2, as described herein, will also be useful for treating a cancer in a patient wherein Wnt2 expression is overexpressed.
  • a cancer status is determined as part of monitoring the effect of surgery (e.g., removal of tumor), the effect of an anti-cancer drug or a therapy administered to a subject diagnosed with a cancer wherein Wnt2 expression is overexpressed.
  • the effect of surgery or an anti-cancer drug or a therapy administered to a subject with cancer may include reoccurrence of cancer, progression of cancer (worsening) and cancer regression (improvement).
  • Wnt2 levels are determined in a biological sample from a subject at various times after surgery or at various times of having been given an anti-cancer drug or a therapy.
  • a higher Wnt2 level at a second time compared to a Wnt2 level at a first time indicates that the cancer in the subject is progressing.
  • a Wnt2 level detected in a biological sample from a subject at a first time (t1; e.g., shortly after surgery) that is higher than the Wnt2 level detected in a comparable biological sample from the same subject taken at a second time (t2; e.g., weeks or months after surgery) may indicate that the cancer in the subject is not reoccurring.
  • t1 e.g., shortly after surgery
  • t2 e.g., weeks or months after surgery
  • Agents of the present invention that are useful for practicing the methods of the present invention include, but are not limited to anti-Wnt2 antibodies and siRNAs of Wnt2. Typically, such agents are capable of (i) binding to Wnt2 mRNA or Wnt2 protein, (ii) interfere with Wnt2 signaling and/or (iii) inhibit binding of Wnt2 protein to other proteins, such as a Frizzled receptor.
  • the agent inhibiting cell proliferation is a siRNA of Wnt2.
  • the present invention provides compositions and methods using RNA interference to modulate Wnt2 expression. These methods and compositions are useful for the treatment of disease, in particular cancer, induction of apoptosis and interfering with Wnt2 signaling.
  • dsRNA double-stranded RNA
  • siRNA small interfering RNA
  • RNA interference has been referred to as “cosuppression”, “post-transcriptional gene silencing”, “sense suppression” and “quelling.”
  • RNAi is an attractive biotechnological tool because it provides a means for knocking out the activity of specific genes. It is particularly useful for knocking out gene expression in species that were not previously considered to be amenable to genetic analysis or manipulation.
  • RNAi is usually described as a post-transcriptional gene-silencing (PTGS) phenomenon in which dsRNAs trigger degradation of homologous mRNA in the cytoplasm.
  • the basic process involves a dsRNA that is processed into shorter units (called short interfering RNAs (siRNAs)) that guide recognition and targeted cleavage of homologous messenger RNA (mRNA).
  • siRNAs short interfering RNAs
  • mRNAi/PTGS can be made in the nucleus or cytoplasm in a number of ways.
  • the processing of dsRNA into siRNAs, which in turn degrade mRNA is a two-step RNA degradation process.
  • the first step involves a dsRNA endonuclease (ribonuclease III-like; RNase III-like) activity that processes dsRNA into sense and antisense RNAs which are 21 to 25 nucleotides (nt) long (i.e., siRNA). In Drosophila, this RNase III-type protein is termed Dicer.
  • the antisense siRNAs produced combine with, and serve as guides for, a different ribonuclease complex called RNA-induced silencing complex (RISC), which cleaves the homologous single-stranded mRNAs. RISC cuts the mRNA approximately in the middle of the region paired with the antisense siRNA, after which the mRNA is further degraded. dsRNAs from different sources can enter the processing pathway leading to RNAi/PTGS.
  • RISC RNA-induced silencing complex
  • the agent for use in the methods of the present invention is a siRNA of Wnt2.
  • siRNA can be used to reduce the expression level of Wnt2.
  • a siRNA of Wnt2 hybridizes to a Wnt2 mRNA and thereby decreases or inhibits production of Wnt2 protein.
  • the siRNA that is introduced into the organism should contain exonic sequences.
  • the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences.
  • the siRNA exhibits greater than 90% or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences less than about 80% identical to the target gene are substantially less effective.
  • the greater homology between the siRNA of Wnt2 and the Wnt2 gene whose expression is to be inhibited the less likely expression of unrelated genes will be affected.
  • the size of the siRNA is important.
  • the present invention relates to siRNA molecules of Wnt2, which are double or single stranded and comprise at least about 19-25 nucleotides, and are able to modulate the gene expression of Wnt2.
  • the siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides in length. More preferably, the siRNA is from about 19 nucleotides to about 25 nucleotides in length.
  • the invention generally features an isolated siRNA molecule of at least 19 nucleotides, having at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of Wnt2, and that reduces the expression of Wnt2 gene or protein.
  • the siRNA molecule has at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of human Wnt2 (GenBank No.NM — 0003391, SEQ ID NO:152).
  • the isolated siRNA molecule has at least one strand that is substantially complementary to 19 to 25 nucleotides comprising nucleotides 714 to 732 of human Wnt2 (GenBank No.NM — 0003391, SEQ ID NO:152).
  • the siRNA nucleic acid sequence is 5′-GAAGATGGGAAGCGCCAAG-3′ (SEQ ID NO:150).
  • the siRNA molecule of Wnt2 includes a sequence that is at least 90% homologous, preferably 95%, 99%, or 100% homologous, to the nucleic acid sequence shown in SEQ ID NO:152. Without undue experimentation and using the disclosure of this invention, it is understood that additional siRNAs of Wnt2 that modulate Wnt2 expression can be designed and used to practice the methods of the invention.
  • the siRNA may also comprise an alteration of one or more nucleotides.
  • Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA).
  • the RNA molecule contains a 3′-hydroxyl group.
  • Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides.
  • the double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
  • Additional modifications of siRNAs e.g., 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation
  • modified siRNAs e.g., 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and
  • RNAi is capable of decreasing the expression of Wnt2 in a cell by at least 10%, 20%, 30%, or 40%, more preferably by at least 50%, 60%, or 70%, and most preferably by at least 75%, 80%, 90%, 95% or more.
  • siRNA into cells can be achieved by methods known in the art, including for example, microinjection, electroporation, or transfection of a vector comprising a nucleic acid from which the siRNA can be transcribed.
  • a siRNA for Wnt2 can be directly introduced into a cell in a form that is capable of binding to Wnt2 mRNA transcripts.
  • the siRNA may be combined or modified with liposomes, poly-L-lysine, lipids, cholesterol, lipofectine or derivatives thereof.
  • Preferred are cholesterol-conjugated siRNA for Wnt2 see, Song et al., Nature Med. 9:347-351 (2003)).
  • the agent used in the methods of the present invention is an anti-Wnt2 antibody as fully described herein.
  • the anti Wnt2 antibody can be a polyclonal or an anti-Wnt2 monoclonal antibody.
  • the methods of the present invention use an anti-Wnt2 monoclonal antibody.
  • Wnt2 protein or cells expressing them or members of the Wnt signaling pathway, e.g., dvl, can also be used in drug screening assays to identify agents that inhibit Wnt signaling.
  • the present invention thus provides novel methods for screening for compositions which inhibit cancer.
  • Assays for Wnt2 signaling can be designed to detect and/or quantify any part of the Wnt2 signaling pathway. For example the ability of an agent to affect intracellular ⁇ -catenin levels or to induce apoptosis in target cells can be measured. Assays suitable for these purposes are described herein.
  • Assays may include those designed to test binding activity of an inhibitor to either the Wnt2 ligand, the Frizzled receptor, or another member of the Wnt2 signaling cascade, e.g., dvl. These assays are particularly useful in identifying agents that modulate Wnt2 activity. Virtually any agent can be tested in such an assay. Such agents include, but are not limited to natural or synthetic polypeptides, antibodies, natural or synthetic small organic molecules, nucleic acids and the like.
  • test agents are based on natural ligands (e.g., Wnt ligands or sFRPs) of the Frizzled receptor.
  • any of the assays for detecting Wnt2 signaling are amenable to high throughput screening.
  • High throughput assays, binding assays and reporter gene assays are similarly well known.
  • U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins
  • U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays)
  • U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
  • high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay.
  • These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems.
  • Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
  • assays useful in the present invention are those designed to test neoplastic phenotypes of cancer cells. These assays include cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cellular proliferation; cell death (apoptosis); cellular transformation; growth factor or serum dependence; tumor specific marker levels; invasiveness into Matrigel; tumor growth and metastasis in vivo; mRNA and protein expression in cells undergoing metastasis, and other characteristics of cancer cells.
  • test agents to inhibit cell growth can also be assessed by introducing the test into an animal model of disease, and assessing the growth of cancer cells in vivo.
  • human tumor cells can be introduced into an immunocompromised animal such as a “nude mouse”.
  • the test agent e.g., a small molecule or an antibody
  • the test agent is administered to the animal and the ability of the tumor cell to form tumors—as assessed by the number and/or size of tumors formed in the animal—is compared to tumor growth in a control animal without the agent.
  • inhibitors of the Wnt2 signaling pathway can comprise nucleic acid molecules that inhibit expression of the target protein in the pathway.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered polypeptides, e.g., dominant negative forms of the protein, in mammalian cells or target tissues, or alternatively, nucleic acids e.g., inhibitors of target protein expression, such as siRNAs or anti-sense RNAs.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • DNA and RNA viruses which have either episomal or integrated genomes after delivery to the cell.
  • RNA viruses which have either episomal or integrated genomes after delivery to the cell.
  • small interfering RNAs are administered.
  • introduction of long dsRNA >30 nt often initiates a potent antiviral response, exemplified by nonspecific inhibition of protein synthesis and RNA degradation.
  • the phenomenon of RNA interference is described and discussed, e.g., in Bass, Nature 411:428-29 (2001); Elbahir et al., Nature 411:494-98 (2001); and Fire et al., Nature 391:806-11 (1998), where methods of making interfering RNA also are discussed.
  • the siRNA inhibitors are less than 100 base pairs, typically 30 bps or shorter, and are made by approaches known in the art.
  • Exemplary siRNAs according to the invention can have up to 29 nucleotides, 25 nucleotides, 22 nucleotides, 21 nucleotides, 20 nucleotides, 15 nucleotides, 10 nucleotides, 5 nucleotides or any integer thereabout or therebetween.
  • inhibitors of Wnt2 expression and agents of the present invention can be used to treat a disease associated with Wnt2 signaling, such as a cancer associated with Wnt2 signaling.
  • the compositions for administration will commonly comprise an agent as fully described herein dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
  • compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges.
  • antibodies when administered orally, should be protected from digestion. This is typically accomplished either by complexing the molecules with a composition to render them resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in an appropriately resistant carrier, such as a liposome or a protection barrier. Means of protecting agents from digestion are well known in the art.
  • compositions containing inhibitors and agents of the invention can be administered for therapeutic or prophylactic treatments.
  • compositions are administered to a patient suffering from a disease (e.g., breast cancer) in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient.
  • prophylactically effective dose An amount of an inhibitor that is capable of preventing or slowing the development of cancer in a patient is referred to as a “prophylactically effective dose.”
  • the particular dose required for a prophylactic treatment will depend upon the medical condition and history of the patient, the particular cancer being prevented, as well as other factors such as age, weight, gender, administration route, efficiency, etc.
  • prophylactic treatments may be used, e.g., in a patient who has previously had cancer to prevent a recurrence of the cancer, or in a patient who is suspected of having a significant likelihood of developing cancer.
  • a “patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications.
  • the patient is a mammal, preferably a primate, and in the most preferred embodiment the patient is human.
  • inhibitors of Wnt signaling may also be used to target or sensitize a cell to other cancer therapeutic agents such as 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.
  • the methods of the invention can be used with radiation therapy and the like.
  • an antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity or antigen-dependent cytotoxicity (ADCC).
  • ADCC antigen-dependent cytotoxicity
  • cancer can be treated by administering to a patient antibodies directed against Frizzled proteins on the surface of cancer cells.
  • Antibody-labeling may activate a co-toxin, localize a toxin payload, or otherwise provide means to locally ablate cells.
  • the antibody is conjugated to an effector moiety.
  • the effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety, such as a cytotoxic agent.
  • the Wnt2 proteins or immunogenic fragments of them can be administered as vaccine compositions to stimulate HTL, CTL, and antibody responses against the endogenous proteins.
  • vaccine compositions can include, e.g., lipidated peptides (see, e.g., Vitiello et al. (1995) J. Clin. Invest. 95:341-349), peptide compositions encapsulated in poly(D,L-lactide-co-glycolide, “PLG”) microspheres (see, e.g., Eldridge et al. (1991) Molec. Immunol. 28:287-294; Alonso et al.
  • Vaccine compositions often include adjuvants.
  • Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis, or Mycobacterium tuberculosis derived proteins.
  • adjuvants are commercially available as, e.g., Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
  • GM-CSF interleukin-2, -7, -12, and other like growth factors
  • Vaccines can be administered as nucleic acid compositions wherein DNA or RNA encoding the Wnt2 olypeptides, or a fragment thereof, is administered to a patient. See, e.g., Wolff et. al. (1990) Science 247:1465-1468; U.S. Pat. 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 (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
  • genes as DNA vaccines are well known, and include placing the desired gene or portion thereof under the control of a regulatable promoter or a tissue-specific promoter for expression in the patient.
  • the gene used for DNA vaccines can encode full-length Wnt2 protein, or may encode portions of the proteins.
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • adjuvant molecules include cytokines that increase the immunogenic response to the polypeptide encoded by the DNA vaccine.
  • the peptides of the invention can be expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode Wnt2 polypeptides or polypeptide fragments. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.
  • BCG Bacille Calmette Guerin
  • BCG vectors are described in Stover et al., (1991) Nature 351:456-460.
  • a wide variety of other vectors useful for therapeutic administration or immunization e.g., adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent. See, e.g., Shata et al., (2000) Mol. Med. Today 6:66-71; Shedlock et al., (2000) J. Leukoc. Biol. 68:793-806; and Hipp et al., (2000) In Vivo 14:571-85.
  • the agents that inhibit Wnt2 signaling can be used in a variety of therapeutic regimens.
  • the agents can be used in methods comprising, but not limited to parenteral (e.g., intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes), topical, oral, local, or transdermal administration. These methods can be used for prophylactic and/or therapeutic treatment.
  • Methods of non-viral delivery of nucleic acids encoding engineered polypeptides of the invention include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
  • lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes
  • the preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • RNA or DNA viral based systems for the delivery of inhibitors of target Wnt pathway proteins, e.g., Dvl, are known in the art.
  • Conventional viral based systems for the delivery of such nucleic acid inhibitors can include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
  • the gene therapy vector be delivered with a high degree of specificity to a particular tissue type, e.g., a lung cancer.
  • a viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
  • Han et al., PNAS 92:9747-9751 (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • FAB fragment-binding protein
  • Fv antibody fragment-binding protein
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • systemic administration e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with inhibitor nucleic acids and re-infused back into the subject organism (e.g., patient).
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique ( 3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • therapeutic nucleic acids can also be administered directly to the organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
  • kits that can be used for the detection of the Wnt2 nucleic acids or proteins disclosed here. Further, kits are provide comprising compositions described herein that allow the user to practice the methods of the invention. In diagnostic and research applications such kits may include any or all of the following: assay reagents, buffers, Wnt2-specific or Frizzled-specific nucleic acids or antibodies, hybridization probes and/or primers, and the like.
  • a therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
  • the kit comprises an agent embracing the specifics as outlined herein, wherein the agent binds Wnt2 protein or Wnt2 nucleic acid, such as mRNA, interferes with Wnt2 signaling, or inhibits binding of Wnt2 protein to other proteins, such as a Frizzled receptor.
  • the kit may further comprise one or more containers for agents and compositions of the present invention and instructions for using the agent to inhibit the proliferation of a cell overexpressing Wnt2, to treat a disease, such as a cancer overexpressing Wnt2, to induce apoptosis in a cell overexpressing Wnt2, to detect a cancer cell overexpressing Wnt2 or to practice any of the methods described herein.
  • a kit comprises a siRNA as shown in SEQ ID NO:150 or a siRNA comprising a nucleic acid of about 19-25 contiguous nucleotides of SEQ ID NO:152, wherein the siRNA binds to Wnt2 mRNA and inhibits translation of Wnt2 mRNA.
  • This kit further comprises one or more containers for agents and compositions of the present invention and instructions for using the siRNA to inhibit the proliferation of a cell overexpressing Wnt2, to treat a disease, such as a cancer overexpressing Wnt2, to induce apoptosis in a cell overexpressing Wnt2, to detect a cancer cell overexpressing Wnt2 or to practice any of the methods described herein.
  • the kit comprises a control siRNA, for example a siRNA comprising a nucleic acid sequence as shown in SEQ ID NO:151.
  • kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • electronic storage media e.g., magnetic discs, tapes, cartridges, chips
  • optical media e.g., CD ROM
  • Such media may include addresses to internet sites that provide such instructional materials.
  • kits for screening for inhibitors of Wnt2 signaling can be prepared from readily available materials and reagents.
  • kits comprise one or more of the following materials: a Wnt2 polypeptide or polynucleotide, reaction tubes and instructions for testing the desired Wnt2 signaling function (e.g., ⁇ -catenin levels).
  • compositions and kits of the present invention embrace the specifics as outlined herein.
  • NSCLC Human non-small-cell lung cancer
  • SAEC normal human small airway epithelial cells
  • NHBE bronchial epithelial cells
  • All cells were cultured at 37° C. in a humid incubator with 5% CO 2 .
  • Fresh lung cancer tissue and adjacent normal lung tissue and fresh mesothelioma tissue and adjacent normal pleural tissue from patients undergoing curative primary resection of their tumors were collected at the time of surgery, and immediately snap-frozen in liquid nitrogen (IRB# H8714-22942-01). These tissue samples were kept at ⁇ 170° C. in a liquid nitrogen freezer before use.
  • TOPFLASH assays of Tcf-dependent transcriptional activity in various cell lines following incubation with anti-Wnt2 monoclonal antibody were performed as described in You et al. Oncogene ( 2004) 23:6170-4). Briefly, cells were plated in six-well plates. After incubation with control or anti Wnt2 monoclonal antibody (10 ⁇ g/ml) for 48 h, the TOPFLASH or FOPFLASH reporter plasmid was transfected transiently into cells as described previously (Uematsu et al., Cancer Res. (2003) 63:4547-51). Tcf-mediated gene transcription was determined by the ration of pTOPFLASH: pFOPFLASH luciferase activity. Each was normalized to luciferase activities of the pRL-TK reporter (cotransfected internalk control). Experiments were performed in triplicate.
  • Annexin-V-PI double staining regime three populations of cells are distinguishable in two color flow cytometry: (a) non-apoptotic cells: annexin-V and PI negative; (b) early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to Annexin V-FITC but excluded propidium iodide; and (c) cells in necrotic or late apoptotic stages were labeled with both Annexin V-FITC and propidium iodide. Then stained cells were immediately analyzed by flow cytometry (FACScan; Decton Dickinson, Franklin Lake, N.J.).
  • TUNEL staining of tumor tissue samples harvested from in vivo experiment was performed using ApopTag Peroxidase In Situ Oligo Ligation Apoptosis Detection Kit (Chemicon International, Temecula, Calif.) according to the manufacturer's protocol.
  • mice Female nude mice, 5-6 weeks old, were injected s.c. with 1 ⁇ 10 7 LOX cells in the dorsal area in a volume of 100 ⁇ l. Three days later, the tumors were uniformly formed and the animals were then intraperitioneally (i.p.) injected with monoclonal anti-Wnt2 antibody, a control monoclonal antibody, or PBS buffer in a volume of 100 ⁇ l as well. Both the monoclonal anti-Wnt2 and the control antibodies were injected at the dose of 250 ⁇ g. Each injection was done twice weekly. Each group consisted of 8 mice.
  • Tumor size was determined at weekly intervals, and tumor volumes were calculated using width (x) and length (y) (x 2 y/2, where x ⁇ y) (Sonoda et al., Cancer Res 61(13):4956-60 (2001)).
  • the CDR sequences of monoclonal antibodies produced by the hybridoma cell lines 17F7.G7, 17F7.E5, 8B11.D2 and 8B11.H6 were sequenced using standard DNA seq techniques. Briefly, PCR was used to amplify the V regions from first strand cDNA.
  • the primer sets comprised a reverse primer in the constant region and an upstream primer hybridizing to the signal peptide region. Multiple upstream primers for both V H and V L were used.
  • DNA sequences for the variable regions of anti-Wnt2 monoclonal antibodies are shown in FIGS. 14-18 and, for example, in SEQ ID NOS: 68, 75, 92, 95, 111, 118, 129, 135, 141 and 144.
  • Anti-Wnt2 monoclonal antibodies were also sequenced directly by protein sequencing. Briefly, protein samples were reduced and Cys was alkylated with iodoacetamide yielding Cys(CAM). Next, the samples were electrophoresed by SDS-PAGE under reducing conditions and electroblotted onto PVDF membrane each yielding light and heavy chain upon staining with Amido Black. Standard protein sequencing techniques were employed. The N-terminal sequence of subclone MG7, for example is shown in SEQ ID NO:146.
  • gene expression profiling was analyzed using a custom array designed to profile the expression of genes involved in and downstream of Wnt signaling with the AmpoLabelling-LPR Kit protocol (GEArray Q Human Wnt Signaling Pathway Gene Array, SuperArray, Frederick, Md.). Briefly, total RNA isolated from the selected tissues was subjected to an RT reaction and cDNA probes were subsequently labeled with Biotin-16-dUTP (Roche), denatured and hybridized overnight in hybridization tubes containing the Wnt specific arrays. Detection was done with a chemiluminescent reaction by using a CDD camera. I mages of spots were converted in numerical data using software provided by the manufacturer. Expression data was matched against the gene list provided by the manufacturer. A representative gene expression array analysis is shown in FIG. 9 .
  • RNA cells were plated into a 6-well plate with media without antibiotics 24 hrs before testing.
  • the ion-exchange HPLC-purified siRNAs (Wnt2 siRNA and non-silencing siRNA control) were purchased from Qiagen-Xeragon (Germantown, Md.).
  • the lyophilized siRNAs were dissolved in annealing buffer and reheated to 95° C. for 1 min followed by 1 hr at 37° C. incubation. We followed the protocol described by Elbashir (Elbashir et al., Methods 26(2):199-213 (2002)). After siRNA transfection, plates were incubated for 3 days at 37° C. before further analysis.
  • siRNA specific for human Wnt2 was derived from the mRNA sequence 5′-GAAGATGGGAAGCGCCAAG-3′ (SEQ ID NO:150) of human Wnt2.
  • the control (nonsilencing) siRNA does not target any known mammalian gene 5′-AATTCTCCGAACGTGTCACGT-3′ (SEQ ID NO:151).
  • Antigenic peptides of human Wnt2 protein were determined using various methods. For example, the EMBOSS (Parker et al., Biochemistry 25:5425-5432 (1986)) finds antigenic sites in proteins. Antigenic peptides were also determined using the method of Kolaskar and Tangaonkar (K&T; FEBS Lett. (1990) 276(1-2):172-4). Both methods led to the identification of similar antigenic peptides of human Wnt2 (Table 1). While most of the antigenic peptide sequences identified can be used to generate antibodies that specifically bind to human Wnt2, some antibodies may also bind to other Wnt proteins due to amino acid sequence homology among various Wnt proteins with human Wnt2.
  • amino acid sequences of SEQ ID NO:42 and SEQ ID NO:44 have homology to human Wnt2B (Wnt13); the amino acid sequence of SEQ ID NO:43 has homology to human Wnt2B (Wnt13), Wnt3, Wnt3A, Wnt5B, and Wnt10A; the amino acid sequence of SEQ ID NO:45 has homology to human Wnt2B (Wnt13) and Wnt4; the amino acid sequence of SEQ ID NO:47 has homology to human Wnt1 and Wnt2B (Wnt13); and the amino acid sequence of SEQ ID NO:53 has homology to human Wnt-2B (Wnt13), Wnt3, and Wnt8B.
  • the antigen used to raise monoclonal anti-Wnt2 antibodies was a synthetic peptide corresponding to amino acid residues 49-63 of the human Wnt2 (Ac-SSQRQLCHRHPDVMR-amide, SEQ ID NO:2). This antigen was chosen bioinformatically based on its hydrophilicity (Parker et al., (1986) Biochemistry 25(19):5425-32), antigenicity (Welling et al., (1985) FEBS Lett. 188(2):215-8), accessibility (Janin, Nature 277:491-2 (1979)), sequence homology (BLAST search), and N-terminal vicinity.
  • the anti-Wnt2 mouse monoclonal antibody (IgG 1 ) was custom-made at Rockland Inc. (Gilbertsville, Pa.).
  • Several hybridoma cell lines were generated of which five were characterized in detail: (1) 17F7.G7 (Subclone A; FIG. 14 ); (2) 17F7.G7 (Subclone B; FIG. 18 ); (3) 17F7.E5 ( FIG. 16 ); (4) 8B11.D2 ( FIG. 15 ); and (5) 8B11.H6 ( FIG. 17 ).
  • Two different light chains were found in 8B11.H6, termed 8B11.H6 (Chain1) and 8B11.H6 (Chain2).
  • each anti-Wnt2 monoclonal antibody analyzed herein comprises the amino acid sequence LVS (SEQ ID NO:58) for V L CDR2.
  • Other CDR sequences are also very similar, such as the V H CDR1 sequences GYTFTDYV (SEQ ID NO:63) and GYTFTTYV (SEQ ID NO:87), differing by one amino acid residue.
  • Monoclonal antibodies were affinity purified by using Protein G and kept at ⁇ 80° C. Seize X mammalian Immunoprecipitation Kit (Pierce Biotechnology, Rockford, Ill.) was used to precipitate Wnt2 protein from cell line extracts according to the manufacture's protocol and followed by Western blotting. Cells were plated in 6-well plates or 10 cm dishes one day before experiments. Then normal media was replaced by media containing antibodies at various concentrations and the cells were incubated at 37° C. in a humid incubator with 5% CO 2 . At various time points the cells were collected using standard protocols for further analysis. Control antibody is mouse IgG 1 MOPC21 from Sigma-Aldrich Co. (St Louis, Mo.). Anti Wnt2 monoclonal antibody 17F7.G7 was used in the experiments described.
  • Wnt2 mRNA expression in human normal and cancer tissues using cDNA arrays was studied. Briefly, multiple RNA microarrays (Clontech) from normal (76), as well as tumor-normal matched (19) human RNA panels were used for the detection of Wnt2 mRNA expression using a Wnt2 cDNA probe. A full-length Wnt2 cDNA probe was radiolabeled and hybridized to the RNA microarrays. In normal human organs, Wnt2 mRNA was expressed in placenta and weakly in fetal lung and normal lung (You et al. Cancer Res. 64:5385-89 (2004)).
  • FIG. 9 A Wnt-related gene expression profile, for example, in mesothelioma cells is shown in FIG. 9 .
  • a monoclonal antibody against a Wnt2 N-terminal peptide was generated (see Example 1). To test whether the monoclonal anti-Wnt2 antibody could bind specifically to the native form of Wnt2 protein in cultured cells, immunoprecipitation with this monoclonal antibody alone or after pre-incubation with blocking peptide (30-fold over the antibody) in cell extracts from three cell lines was performed. The C57Wnt2 cell line served as a positive control. NSCLC (A549) and melanoma (LOX) cell lines were also tested. In C57Wnt2, A549 and LOX cells, Wnt2 protein was precipitated by the monoclonal anti-Wnt2 antibody.
  • Wnt2 protein expression in numerous human cancer cell lines was tested using this monoclonal antibody; including four human NSCLC cell lines (A549, H1299, H1703, and H460), one normal mesothelial cell line (LP9; FIG. 10 ) and eight malignant mesothelioma cell lines (Met5a, LARk1A, REN, H290, H2052, MS-1, H513, and H28, FIG. 10 ), four human melanoma cell lines (LOX, FEM, FEMX, and SK-Mel-2), two breast cancer cell lines (HuL100 and MCF-7), as well as two colon cancer cell lines (HCT116 and SW480).
  • SAEC Small airway epithelial cells
  • NHBE normal primary human bronchial epithelial cells
  • Wnt2 protein expression was found in all of the cancer cell lines tested. Wnt2 expression was not observed in the two primary normal lung cells (SAEC and NHBE).
  • 7 out of 8 freshly resected NSCLC tissues had increased expression of Wnt2 protein compared with autologous matched normal lung tissue controls (data not shown).
  • 5 out of 5 freshly resected pleura mesothelioma from patients showed Wnt2 protein expression, while Wnt2 protein was not detected in autologous matched normal pleura ( FIG. 10 ).
  • Anti-Wnt2 Monoclonal Antibody Mediates Cytotoxicity/Apoptosis in Primary Cell Cultures from Cancer Patients
  • Anti-Wnt2 monoclonal antibody was used to treat a number of human cancer cell lines including four human NSCLC cell lines (A549, H1299, H1703, and H460), five malignant pleural mesothelioma cell lines (H290, H2052, MS-1, H513, and H28), four human melanoma cell lines (LOX, FEM, FEMX, and SK-Mel-2), two breast cancer cell lines (HuL100 and MCF-7), as well as two colon cancer cell lines (HCT116 and SW480). After 3-5 days of incubation significant cell death in all these cell lines (over 90% cell death at 10.0 ⁇ g/ml of the antibody, P ⁇ 0.005) was observed (Table 5).
  • FIG. 11 cytotoxic effects of anti-Wnt2 monoclonal antibody in hepatoma cell lines HepG2 and Hep3B were observed (data not shown).
  • FIG. 1 A representative example of this analysis using A549 is shown in FIG. 1 .
  • FIG. 6 A similar experiment in melanoma cells is shown in FIG. 6 .
  • Anti-Wnt2 Monoclonal Antibody Inhibits Wnt Signaling and Induces Apoptosis Through Release of Cytochrome c, Down-regulation of Survivin and Activation of Caspase-3
  • TOPFLASH assay P ⁇ 0.01
  • c-Myc and fibronectin were also down-regulated after anti-Wnt2 monoclonal antibody treatment of LOX cells ⁇ FIGURE? ⁇ and of primary tumor cultures freshly made from patients with malignant melanoma ( FIG. 6 ).
  • RNA interference was carried out by following the protocol described by Elbashir et al. (Elbashir et al., Methods 26(2): 199-213 (2002)) and Wnt2 targeted small interfering RNA (siRNA) was used to study the effect of Wnt2 mRNA silencing. Similar to the monoclonal anti-Wnt2 antibody, treatment with Wnt2 siRNA for 3-5 days induced apoptosis in all cancer cell lines expressing Wnt2 (See, FIG. 3 for A549 cells; FIG. 7 for LOX and FEMX cells; and FIG. 12 for MS1, H28 and LRK1A cells).
  • Anti-Wnt2 Monoclonal Antibody Suppresses Tumor Growth In Vivo
  • mice were inoculated through the tail vein into athymic nude mice at 3 million cells/per mouse.
  • Ten mice were then treated with 250 ⁇ g of the monoclonal anti-Wnt2 antibody or PBS control via intraperitioneal (i.p.) injection twice a week for four weeks. Mice were sacrificed one week after. Lungs were collected surgically and weighed. Results were analyzed with Student's t-test. A difference in lung weight was observed between anti Wnt2 monoclonal antibody treated mice and untreated control mice (P ⁇ 0.0418; FIG. 4 ).
  • malignant melanoma LOX cells were injected s.c. into nude mice.
  • the animals were then treated with 250 ⁇ g of the anti-Wnt2 monoclonal antibody, control antibody, or 100 ⁇ l PBS control via i.p. injections twice a week.
  • the monoclonal anti-Wnt2 antibody significantly inhibited tumor growth versus control ( FIG. 8 ). Suppression of tumor growth was seen when the anti-Wnt2 monoclonal antibody injection was started after the tumors were already established (three days after tumor cell inoculation) (P ⁇ 0.005; FIGS. 8 a and 8 b ).
  • the tumor tissues were harvested and analyzed via TUNEL staining and apoptotic cells were noticed in those tumor tissues treated with the anti-Wnt2 monoclonal antibody.
  • Anti-Wnt2 Antibody-Induced Apoptosis is a Fast Process and Dose Dependent
  • Anti-Wnt2 Antibody-Induced Apoptosis is Associated with Releasing of Smac/Diablo and Cytochrome C from Mitochondria to the Cytosol and JNK Activation
  • NSCLC non small-cell lung cancer
  • Dvl expression and function was analyzed in order to evaluate the function of Wnt2 signaling in NSCLC.
  • Eight NSCLC fresh tumors (four squamous cell and four adenocarcinomas) and their autologous matched normal lung tissue were obtained from patients undergoing resection of their tumors as part of their treatment for early stage I NSCLC. Patients had not received any prior treatment, e.g., chemotherapy.
  • Western blot analysis of these samples showed that in 75% (three of four squamous cell carcinomas and three of four adenocarcinomas) of all cancer cells tested, Dvl-3 was overexpressed while the corresponding matched normal microdissected lung tissues failed to show expression of Dvl-3.
  • seven of eight NSCLC tumors with Dvl-3 overexpression showed higher expression of Wnt2 by western blot analysis. Expression of Dvl-1 or Dvl-2 was not detected.
  • an anti-Wnt2 monoclonal antibody was used to further investigate the specificity of the effect of anti-Wnt2 antibodies.
  • the anti-Wnt2 monoclonal antibody was able to induce apoptosis in human cancer cell lines that over-express Wnt2 protein, e.g., human lung cancer cell line. Similar to the results obtained from polyclonal antibody study, both dvl and cytosolic ⁇ catenin proteins were decreased after the anti-Wnt2 monoclonal antibody treatment in these tumor cells.
  • the anti-Wnt2 monoclonal antibody showed much higher specificity than the anti-Wnt2 polyclonal antibody, e.g., the anti-Wnt2 monoclonal induces apoptosis only in the tumor cells that over-express Wnt2 protein (A549, LOX and FEMX), and has no detectable effect in the tumor cells that express Wnt2 protein; the anti-Wnt-1 polyclonal antibody induces apoptotic cell death in the tumor cells that over-express Wnt2. Taken together, these data indicate that the anti-Wnt2 antibody treatment can induce tumor-specific apoptosis and down-regulate the Wnt-dvl- ⁇ catenin signaling pathway in human cancer cells.
  • Wnt signal activates two distinct pathways: the canonical pathway (i.e., ⁇ catenin pathway) and the JNK pathway.
  • Dishevelled protein has three highly conserved domains, DIX, PDZ, and DEP. Among them, the DIX and PDZ domains are necessary for the canonical signaling pathway while the DEP domain is important for the activation of JNK pathway. It has been suggested that the activation of JNK plays a critical role in initiating apoptosis (Wang, C. Y. et al., Mol Cell Biol 19(9):5923-9 (1999)). Recently, Chen et al.
  • siRNA-mediated inhibition of Dvl expression in NSCLC cells decreased ⁇ -catenin-mediated Tcf transcription, which further supports that Dvl overexpression is important to the canonical Wnt/B-catenin pathway in some lung cancer cells.
  • Inhibition of Dvl also suppressed cell growth and colony formation in NSCLC cells, which indicates that aberrant upstream events in Wnt signaling is related to tumorigenesis in NSCLC.
  • Smac/Diablo second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI
  • Smac/Diablo second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI
  • wnt antibodies may not only induce directly apoptosis in cancer cell that overexpress wnt proteins, but also release potentially drug resistance by restoring normal apoptotic machinery back to these tumor cells.
  • the basis for drug resistance in tumor cells is most likely the disruption of apoptosis.
  • Over expression of Survivin an inhibitor of apoptosis, is a common feature of most human cancers. It has been shown that targeting of survivin increases the sensitivity of tumor cells to cytotoxic drugs (Grossman, D. et al., Proc Natl Acad Sci USA 98(2):635-40 (2001)). It has been shown that antisense survivin is sufficient to cause apoptosis in human mesothelioma cells. Moreover, a synergistic effect between antisense surviving and chemotherapy has also been reported.
  • fz frizzled
  • LRP LRP-related protein
  • mutant and/or truncated forms of all Fz receptors and/or LRP co-receptors extracellular, transmembrane, and/or intracellular domains
  • the cancer types that we tested include breast cancer, colon cancer, prostate cancer, lung cancer, mesothelioma, and sarcoma.
  • the genetic alterations mentioned above include chromosomal deletion (homozygous or heterozygous), chromosomal translocation, chromosomal breaks, chromosomal inversions, internal small deletions, insertions, and point mutations.
  • Fz receptors and/or LRP co-receptors result in constitutive signaling regardless presence of Wnt ligands, which in turn result in constitutive downstream transcriptional activities in cancers.
  • Wnt ligands Wnt ligands
  • mutant and/or truncated forms of Fz receptors and/or LRP co-receptors for the Wnt signaling pathway are cancer specific. They have very strong potential to be used as targets for developing therapeutic drugs (e.g., small molecules, chemical compounds, antibodies, antisense-oligos or RNAi as discussed above). These drugs are able to target cancers only, but not normal cells. Thus, this invention will be of great help in therapeutic strategies for treatment of a number of cancers as noted above, including colon cancer, breast cancer, lung cancer, e.g., NSCLC, mesothelioma and sarcoma, and the like.
  • therapeutic drugs e.g., small molecules, chemical compounds, antibodies, antisense-oligos or RNAi as discussed above. These drugs are able to target cancers only, but not normal cells.
  • this invention will be of great help in therapeutic strategies for treatment of a number of cancers as noted above, including colon cancer, breast cancer, lung cancer, e.g., NSCLC

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CA2566498A1 (fr) 2005-12-08
EP1753880A4 (fr) 2010-07-14
US7959923B2 (en) 2011-06-14
WO2005116236A3 (fr) 2006-11-09
WO2005116236A2 (fr) 2005-12-08
US20090202539A1 (en) 2009-08-13
EP1753880A2 (fr) 2007-02-21
JP2007536938A (ja) 2007-12-20

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