KR20140018411A - Wnt proteins and detection and treatment of cancer - Google Patents

Abstract

The present invention provides methods for diagnosing and treating cancer using Wnt and FZD proteins, genes, FZD and Wnt specific antibodies and probes, and screening whether test compounds possess efficacy in treating cancer. The present invention also discloses compounds useful for the treatment of cancer, for example liver cancer.

Description

WNT protein and cancer detection and treatment {WNT PROTEINS AND DETECTION AND TREATMENT OF CANCER}

Statement on federally funded research

The present invention has been approved by the National Institutes of Health under government support, and the approval numbers are CA035711 and AA002666. The government reserves certain rights in the invention.

Cross-references to related applications

This application continues to apply in part of International Patent Application No. PCT/US05/000267 (filed January 5, 2005), claiming priority to US Provisional Specification Applicant No. 60/612,098 (filed September 21, 2004) And both of these documents are incorporated herein by themselves for reference.

Technical field

The present invention relates to the detection and treatment of liver cancer.

Liver cancer (HCC) is the most common malignant tumor in the liver. Although viral etiological factors have been identified, most of the molecular mechanisms involved in tumor progression in the process of developing liver cancer are unknown. The frizzled family of proteins consists of 10 or more seven-transmembrane proteins that act as receptors for Wnt proteins. The Wnt/frizzled signaling network influences a variety of biological processes, from cell fate determination to cell mobility and proliferation.

β-catenin is a multifactorial protein that plays a role in cell-cell adhesion, including strongly binding catherin and α-catenin to the actin cytoskeleton. In the absence of Wnt/frizzled signaling, β-catenin is phosphorylated by interaction with glycogen synthase kinase (GSK)-3β, along with axin and adenomatous polyposis coli protein (APC). To form a complex. Ultimately, β-catenin is targeted for degradation by the ubiquitin-proteasome system. Conversely, when Wnt ligand binds to its frizzled receptor, it inhibits GSK-3β enzyme activity, thereby stabilizing β-catenin in cells. Thereafter, β-catenin binds to high mobility group domain factors such as Tcf/Lef and moves into the nucleus. This complex upregulates the transcription of genes related to growth regulation and cell migration.

1A is a two-dimensional SDS-PAGE gel showing the pattern of fractionated heparan sulfate proteoglycans (HSPG) derived from Huh7 cells after treatment with heparin (Hep+) or without treatment (Hep-) in Huh7 cells as silver. It is a picture showing the dyeing result. The 0.25M NaCl fraction obtained from heparin affinity chromatography was separated on a 1-dimensional nonlinear IPG gel with a pH of 3-10 and a 2-dimensional NuPAGE gel with a 4-12% gradient. As a result of observing the protein spot (circled part; hereinafter referred to as "first spot") obtained from the heparin-untreated fraction, it was found that the expression was increased.
1B is a mass spectrum result of a trypsin-treated peptide derived from the first spot. The first spot was cut out, bleached, and then digested with trypsin. Peptide mass was analyzed on a PBSII instrument. The indicated peak represents a peptide that matches the calculated mass of the human Wnt11 peptide.
1C is an agarose gel photograph showing the results of detecting Wnt ligand mRNA of various hepatocellular carcinoma cell lines using RT-PCR. Wnt3 mRNA was detected in all HCC cell lines, and Wnt11 mRNA was detected in 3 HCC cell lines (except for FOCUS cells). No more Wnt mRNA was detected by RT-PCR.
2A is a bar graph showing the results of measuring Wnt3 mRNA levels in HCC cell lines by qRT-PCR. The 18S rRNA level was used as an internal control, and the mRNA levels of Wnt3 and Wnt11 were expressed as the number of copies per ^{10 9 18S rRNAs.} The expression level (mean ± SE) of Wnt3 mRNA in HCC cell lines was 370.0 ± 10.3 for HepG2, 381.3 ± 12.7 for Hep3B, 95.2 ± 6.3 for Huh7, and 210.4 ± 9.5 copies per ^{10 9 18S rRNAs.} Corresponds to.
Figure 2b is a bar graph showing the results of measuring the Wnt11 mRNA level in the HCC cell line by qRT-PCR. The 18S rRNA level was used as an internal control, and the mRNA level of Wnt11 was expressed as the number of copies per ^{10 9 18S rRNAs.} The Wnt11 mRNA expression level was 8,499.3 ± 845.0 for HepG2, 290.9 ± 40.1 for Hep3B, and 3.57 ± 0.2 copies per ^{10 9 18S rRNAs in Huh7 cells.} Wnt11 mRNA could not be detected by qRT-PCR in focus cells, and this was the same in the case of conventional RT-PCR.
3A is a bar graph showing the expression of Wnt3 mRNA in human HCC tissue. mRNA levels were measured by qRT-PCR. White bars represent mRNA levels in normal liver tissue. Black bars represent mRNA levels in HCC tissue. Gray bars represent the corresponding mRNA levels in the peri-tumor tissue. The experiment was conducted twice, and the data are expressed as mean ± SD. In 77% of HCC and 59% of peri-tumor tissue, the expression level of Wnt3 mRNA increased by a level above the mean ± 3SD value in normal liver tissue. In 71% of HCC, the Wnt3 mRNA expression level was higher than the corresponding Wnt3 mRNA expression level in the peri-tumor tissue.
3B is a bar graph showing the expression of Wnt11 mRNA in human HCC tissue. mRNA levels were measured by qRT-PCR. White bars represent mRNA levels in normal liver tissue. Black bars represent mRNA levels in HCC tissue. Gray bars represent the corresponding mRNA levels in the peri-tumor tissue. The experiment was conducted twice, and the data are expressed as mean ± SD. In 41% of HCC tissues, the expression level was reduced below the minimum cut-off level in normal liver tissue, whereas not in the peri-tumor tissue (P = 0.0036, Fisher fit test). In 65% of the paired samples, it was found that the level of Wnt11 mRNA expression in the tumor was decreased when compared to the corresponding tissue around the tumor.
3C is a bar graph showing the expression of FZD7 mRNA in human HCC tissue. mRNA levels were measured by qRT-PCR. White bars represent mRNA levels in normal liver tissue. Black bars represent mRNA levels in HCC tissue. Gray bars represent the corresponding mRNA levels in the peri-tumor tissue. The experiment was conducted twice, and the data are expressed as mean ± SD. In 59% of both HCC and peri-tumor tissues, FZD7 mRNA expression was increased compared to that of normal liver tissue. In 71% of the paired samples, it was found that the level of FZD7 mRNA expression in the tumor was increased when compared to the corresponding peri-tumor tissue (P = 0.031, Wilcoxon rank sum test).
4A-4D are photographs of immunohistochemically stained human HCC and peri-tumor tissue (400 times magnification). Fig. 4a is a negative control, and Fig. 4b shows a tissue around the tumor stained with β-catenin. Liver cells are usually membrane stained. Figure 4c shows β-catenin staining HCC tissue, in this case, β-catenin was accumulated in the nucleus. It is worth paying attention to the results of nuclear staining of β-catenin and cytoplasmic staining with increased concentration. 4D shows HCC tissues in which β-catenin is not accumulated in the nucleus but accumulated in the cytoplasm.
5A shows the results of a plurality of Western blots showing the effect of Wnt3 plasmid transfection on T-cell factor (Tcf) transcriptional activity in HCC cell lines. Focus, Huh7 and Hep3B cells were co-transfected with myc -tagged Wnt3 plasmid or pcDNA3.1/ myc- His A plasmid, pSUPER8xTOPFLASH or pSUPER8xFOPFLASH, and β-galactosidase plasmid. After 24 hours of transfection, serum was removed from the cell culture solution, and the cells were cultured for 24 hours, and then 1% FBS MEM was added. Cells were collected 2 hours and 24 hours after the addition as described above, and a luciferase assay was performed, followed by Western blot analysis for Wnt3 and β-catenin. Western blot analysis with rabbit polyclonal anti-Wnt3 antibody revealed that the Wnt3 protein was increased after transfection. The specificity of the polyclonal anti-Wnt3 antibody was confirmed using a monoclonal anti-myc antibody. It should be noted that the intracellular β-catenin level was increased in the focus cells. Hsp90 protein was used as an internal loading control.
5B is a bar graph showing changes in Tcf transcriptional activity in HCC cell lines after transfection with Wnt3 plasmid. Tcf transcriptional activity was increased three-fold in focal cells after transfection as compared with the case of focal cells transfected with the control plasmid. Tcf transcriptional activity was slightly decreased in Huh7 cells after transfection, but did not change in Hep3B cells. White bars represent cells transfected with the control plasmid (pcDNA), and black bars represent cells transfected with the Wnt3 plasmid.
6A is a bar graph showing the influence of anti-Wnt3 antibodies on Tcf transcriptional activity in HCC cell lines. HCC cells were inoculated into 12-well plates and transfected with pSUPER8xTOPFLASH or pSUPER8xFOPFLASH with β-galactosidase plasmid. After 24 hours with the serum subtracted from the cell culture, the cells were mixed with 1% FBS MEM containing either an anti-Wnt3 antibody (Wnt3-Ab; black bar) or a control antibody (10 μg/ml). After incubation, cells were collected again 24 hours later. Normal rabbit IgG was used as a control antibody (C-Ab; white bar). When treated with polyclonal anti-Wnt3 antibody, Tcf transcription activity decreased by 60% in Huh7, 26% in Hep3B cells, and 40% in focal cells.
6B is a bar graph showing the influence of siRNA on endogenous Wnt3 mRNA expression. Control siRNA and Wnt3 siRNA (WNT3-3) were transfected into HCC cells at a concentration of 10 or 100 nM. Cells were collected 48 hours after transfection, and then, the level of Wnt3 mRNA expression was measured using qRT-PCR. siRNA Wnt3-3 reduced mRNA levels by an average of 50-60% at a concentration of 100 nM in all three cell lines. 6B shows such an effect in Huh7 cells.
6C is a bar graph showing the influence of Wnt3 siRNA on Tcf transcription activity or HCC cell line. Wnt3 siRNA or control siRNA were co-transfected under conditions in which the Tcf reporter was present at the indicated concentration (nM). Tcf transcriptional activity decreased by 48.5% for Huh7, 33% for Hep3B, and 43.5% for focus cells.
7 is a photograph of a cell culture solution showing that the wound healing of focus cells treated with an anti-Wnt3 antibody is delayed. Focus cells were plated on 6 well plates. The confluent monolayer cells were wounded with the tip of a 200 μl sterile plastic micropipette. Thereafter, the cells were treated with a medium containing either anti-Wnt3 Ab or rabbit IgG (control antibody (C Ab); 10 μg/ml), and photographs were taken at different time points. It was found that the anti-Wnt3 Ab-treated focal cells had a delay in wound healing. This effect was strongest after 24 hours. After 24 hours, most of the wounds were covered with migratory cells and/or proliferative cells among cells treated with C Ab, whereas focal cells treated with anti-Wnt3 Ab did not.
8A-8E show representative FZD7, FZD8, Wnt3, Wnt8b and Wnt11 human and mouse amino acid sequences, including putative binding motifs.

Summary of the invention

In the present invention, in part, Wnt3, 8b and 11 are HCC, for example, in the hepatitis B virus (HBV) related HCC, in which the mRNA and protein are generally expressed at an excessive level, a ligand of Frizzled 7 It is based on the fact. Liver cancer cells that overexpress frizzled 7 have strong mobility and migration. Such overexpression is an early phenomenon in the process of hepatocyte transformation consisting of a number of stages, and frizzled 7 and Wnt 3, 8b and 11 are considered to be novel target molecules for the treatment of liver cancer.

Therefore, in one aspect, the present invention provides a method of identifying an anticancer agent. The method comprises the steps of selecting a test compound that binds to a polypeptide comprising the amino acid sequence of the Wnt3, Wnt8b or Wnt11 protein or FZD-binding fragment thereof; Optionally, this test compound is capable of (i) reducing Wnt/FZD7 signaling in cells; (ii) can reduce hepatic cell mobility; (iii) whether it can reduce the accumulation of β-catenin in liver cancer cells; Or (iv) determining whether it is possible to treat liver cancer in vitro or in vivo, wherein the test compound having the efficacy of at least one of (i) to (iv) is an anticancer agent. In some embodiments, selecting the test compound comprises providing a polypeptide comprising the amino acid sequence of a Wnt3, Wnt8b or Wnt11 polypeptide or an FZD binding fragment thereof; Contacting the polypeptide with the test compound; Determining whether binding between the polypeptide and the test compound; And selecting the test compound when the test compound binds to the polypeptide. The polypeptide to which the test compound binds may be (i) a naturally occurring polypeptide, (ii) a recombinant polypeptide, (iii) a polypeptide expressed on the cell surface, or (iv) an isolated polypeptide. When the polypeptide comprises the amino acid sequence of the Wnt3 protein, the polypeptide may comprise any one of SEQ ID NOs: 8 to 12. When the polypeptide comprises the amino acid sequence of the Wnt 8b protein, the polypeptide may comprise any one of SEQ ID NOs: 15 to 19. When the polypeptide comprises the amino acid sequence of the Wnt11 protein, the polypeptide may comprise any one of SEQ ID NOs: 22-26. In certain embodiments, the polypeptide comprises any one of SEQ ID NOs: 7-27 and one or more non-Wnt sequences. The test compound can be selected from the group consisting of polypeptides, ribonucleic acids, small molecules (eg, small organic molecules) and deoxyribonucleic acids.

Examples of anticancer agents identified by the method for identifying anticancer agents described herein include anti-Wnt antibodies such as monoclonal antibodies, FZD7 receptors, Wnt-binding fragments of FZD7 receptors, and other Wnt-binding compounds. Anticancer agents identified by this method can be used to treat cancer, for example, liver cancer. In addition, anticancer agents identified by the present method can be used to treat liver cancer in patients and to manufacture drugs that reduce liver cancer cell mobility.

In another aspect, the present invention includes a method of identifying a candidate anticancer agent. The method comprises (a) (i) a FZD polypeptide (e.g., FZD7 polypeptide) or a fragment thereof, and (ii) a first retains Wnt (e.g., Wnt 3, 8b or 11)-binding ability. Providing a polypeptide; (b) (i) providing a second polypeptide comprising a Wnt polypeptide (e.g., Wnt3, 8b or 11 polypeptide) or a fragment thereof, and (ii) retaining the ability to bind FZD (e.g., FZD7). ; (c) contacting the first polypeptide and the second polypeptide in the presence of a test compound; And (d) comparing the level of binding between the first polypeptide and the second polypeptide in the presence of the test compound to the level of binding in the absence of the test compound, wherein the level of binding in the presence of the test compound is this test. Lower than the level of binding in the absence of the compound means that the test compound is a candidate anticancer agent. The present method includes (e) whether this candidate anticancer agent can (i) reduce Wnt/FZD7 signaling in cells, (ii) reduce cancer cell mobility, and (iii) accumulate β-catenin in cancer cells. It may further comprise the step of determining whether it is possible to reduce the risk or (iv) can treat cancer cells in vitro or in vivo, wherein the at least one characteristic of (i) to (iv) is Candidate to have is anticancer agent The test compound may be selected from the group consisting of polypeptides, ribonucleic acids, small molecules (eg, small organic molecules), and deoxyribonucleic acids. The Wnt polypeptide may comprise the sequence of SEQ ID NOs: 8-12, SEQ ID NOs: 15-19, and/or SEQ ID NOs: 22-26, for example. The FZD polypeptide may comprise the sequence of SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3.

In certain embodiments, the first polypeptide comprises a FZD polypeptide (e.g., FZD7 polypeptide) fused to (i) a transcriptional activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor. Protein; The second polypeptide is a second fusion protein comprising (i) a transcriptional activation domain of a transcription factor or (ii) a Wnt polypeptide fused to a DNA-binding domain of a transcription factor (e.g., Wnt 3, 8b or 11 polypeptide). As, the Wnt polypeptide is fused to a domain different from the domain fused to the Wnt polypeptide; The binding of the first polypeptide and the second polypeptide is determined by reconstitution of the transcription factor.

Anticancer agents (and/or candidate anticancer agents) identified by the methods described herein can be used in the treatment of cancer, such as liver cancer. In addition, anticancer agents (and/or candidate anticancer agents) identified by the methods described herein can be used to prepare drugs for the treatment of cancer, such as liver cancer.

In another aspect, the present invention provides a method of determining whether a cell (eg, a liver cell) is a cancer cell or is at risk of developing the cancer cell. The method comprises the steps of: (a) providing a test cell (eg, a liver cell); (b) whether the intracellular expression level of FZD7 and/or Wnt3 is higher than that of the control cells, and/or whether the intracellular expression level of FZD8 and/or Wnt11 is lower than that of the control cells. Measuring whether or not; And (c) the FZD7 and/or Wnt3 expression level of the test cell is higher than the FZD7 and/or Wnt3 expression level of the control cell and/or the FZD8 and/or Wnt11 expression level of the test cell is FZD8 and the control cell. And/or if it is lower than the Wnt11 expression level, classifying the test cell as (i) cancer cells or (ii) cells at risk of developing cancer cells. Where the method comprises measuring the level of FZD7 and/or Wnt3 expression in the cell, the method comprises (c) the FZD8 and/or Wnt11 expression level of the test cell is higher than that of the control cell. It may further comprise determining whether it is low, wherein a lower level of expression of FZD8 and/or Wnt11 means that the test cell is a cancer cell or is at risk of developing cancer cells. When the method comprises measuring the level of FZD8 and/or Wnt11 expression in the cell, the method comprises (c) the FZD7 and/or Wnt3 expression level of the test cell is higher than that of the control cell. It may further comprise determining whether it is high, wherein a higher expression level of FZD7 and/or Wnt3 means that the test cell is a cancer cell or is at risk of developing a cancer cell.

In a further aspect, the present invention provides a method of determining whether a patient has developed cancer or is at risk of developing cancer, for example, whether a test tissue sample is derived from a patient who has developed cancer or is at risk of developing cancer. do. The method comprises the steps of: (a) providing a test tissue sample obtained from a patient (eg, liver tissue, eg, tumorous or peritumorous liver tissue); And (b) whether the expression level of FZD7 and/or Wnt3 in the test tissue sample is higher than the expression level of FZD7 and/or Wnt3 in the comparison tissue sample obtained from a healthy individual, and/or FZD8 in the comparison tissue sample obtained from a healthy individual, and / Or determining whether the expression level of FZD8 and/or Wnt11 in the test tissue sample is lower than the expression level of Wnt11, wherein the expression level of FZD7 and/or Wnt3 in the test tissue sample is higher and/or higher. Or, the lower expression level of FZD8 and/or Wnt11 in the test tissue sample is an indicator that the sample is derived from a patient with or at risk of developing cancer. When the method comprises the step of measuring the expression level of FZD7 and/or Wnt3, the method comprises (c) the expression level of FZD8 and/or Wnt11 in the test tissue sample is FZD8 and/or Wnt11 in a tissue sample obtained from a healthy individual. It may further include determining whether the expression level of FZD8 and/or Wnt11 is lower than the expression level of, wherein the sample is derived from a patient with or at risk of developing cancer. It becomes an indicator that it has become. Where the method includes measuring the expression level of FZD8 and/or Wnt11, the method comprises (c) the expression level of FZD7 and/or Wnt3 in the test tissue sample is FZD7 and/or in a tissue sample obtained from a healthy individual. The step of determining whether the expression level of Wnt3 is higher than that of Wnt3 may be further included, wherein the higher expression level of FZD7 and/or Wnt3 is an indicator that the patient has developed or is at risk of developing cancer.

In any of the methods described herein, the step of measuring the expression level of FZD7, FZD8, Wnt3 or Wnt11 comprises determining the amount of the mRNA of FZD7, FZD8, Wnt3 or Wnt11 in the cell, e.g., Northern blot assay or RT. -May include the step of measuring using a PCR assay. In another embodiment, the step of measuring the expression level is the amount of the FZD7, FZD8, Wnt3 or Wnt11 protein in the cell, e.g., using an anti-Wnt antibody that binds to an antibody such as SEQ ID NO: 7 to SEQ ID NO: 80. It may include the step of measuring.

In another aspect, the present invention includes a method of treating cancer (eg, liver cancer) in a cancer patient. The method comprises administering to the patient an effective amount of a compound, i.e., a compound that reduces Wnt/FZD7 signaling in the patient's FZD7 expressing cells and which is not even lethal to the FZD7 expressing cells. In one embodiment, the compound is a compound that decreases the expression of FZD7 and/or Wnt3 in a patient and/or increases the expression of Wnt11 in a patient. In another embodiment, the compound is a compound that binds to Wnt3, Wnt8b, Wnt11, FZD7 or FZD8 in a patient. Examples of these compounds include antisense oligonucleotides, double-stranded RNA (dsRNA) comprising a nucleotide sequence that hybridizes to a Wnt nucleotide sequence under physiological conditions, an isolated FZD7 receptor or a Wnt3 binding fragment thereof, a Wnt polypeptide (e.g., Wnt11 polypeptide) Or a genetic construct encoding a truncated form of FZD7 (e.g., a form of FZD7 lacking the intracellular domain and/or transmembrane domain of FZD7), and/or an anti-FZD and/or anti-Wnt antibody (e.g., Anti-Wnt3 antibody). The compound can be administered by any route, such as administration to the liver of a patient. In certain embodiments, the compound is an antibody that binds to the sequence of SEQ ID NO: 7 or SEQ ID NO: 80. In another embodiment, the compound is an siRNA comprising the sequence of SEQ ID NO: 81, 82 or 83.

In another aspect, the present invention includes a method of reducing the mobility of cancer cells (eg, liver cancer cells). The method involves administering to the cell an effective amount of a compound, i.e., a compound that reduces intracellular Wnt/FZD7 signaling and is not fatal to the cell. In one embodiment, the compound is a compound that decreases the expression of FZD7 and/or Wnt3 in a patient and/or increases the expression of Wnt11 in a patient. In another embodiment, the compound is a compound that binds to Wnt3, Wnt8b, Wnt11, FZD7 or FZD8 in a patient. Examples of these compounds include antisense oligonucleotides, double-stranded RNA (dsRNA) comprising a nucleotide sequence that hybridizes to a Wnt nucleotide sequence under physiological conditions, an isolated FZD7 receptor or a Wnt3 binding fragment thereof, a Wnt polypeptide (e.g., Wnt11 polypeptide) or Genetic constructs encoding the truncated form of FZD7 (e.g., a form of FZD7 lacking the intracellular domain of FZD7 and/or the transmembrane domain), and/or anti-FZD and/or anti-Wnt antibodies (e.g., anti Wnt3 antibody). This compound can be administered by any route, for example by administration to the liver of a patient. In certain embodiments, the subject compound is an antibody that binds to the sequence of SEQ ID NO: 7 or SEQ ID NO: 80. In another embodiment, the subject compound is an siRNA comprising the sequence of SEQ ID NO: 81, 82 or 83.

In another aspect, the present invention includes the use of a compound that reduces Wnt/FZD7 signaling in FZD7 expressing cells in the process of manufacturing (i) a drug for treating liver cancer or (ii) a drug that reduces the mobility of liver cancer cells. . Optionally, the drug is not lethal to FZD7 expressing cells. In one embodiment, the compound is a compound that decreases the expression of FZD7 and/or Wnt3 in a patient and/or increases the expression of Wnt11 in a patient. In another embodiment, the compound is a compound that binds to Wnt3, Wnt8b, Wnt11, FZD7 or FZD8 in a patient. Examples of these compounds include antisense oligonucleotides, double-stranded RNA (dsRNA) comprising a nucleotide sequence that hybridizes to a Wnt nucleotide sequence under physiological conditions, an isolated FZD7 receptor or a Wnt3 binding fragment thereof, a Wnt polypeptide (e.g., Wnt11 polypeptide). Or a genetic construct encoding a truncated form of FZD7 (e.g., a form of FZD7 lacking the intracellular domain and/or transmembrane domain of FZD7), and/or an anti-FZD and/or anti-Wnt antibody (e.g., Anti-Wnt3 antibody).

In any aspect, the invention includes an anti-Wnt antibody, e.g., an anti-Wnt3 antibody, e.g., an antibody that binds to SEQ ID NO: 7 or the amino acid sequence LRAKYSLFKPPTERDL (SEQ ID NO: 80). The antibody may be included in a pharmaceutical composition suitable for administration to a patient.

In another aspect, the present invention includes the use of any of the compounds disclosed herein in the manufacture of a pharmaceutical composition for the treatment or prevention of a condition disclosed herein, such as cancer, such as liver cancer. The composition can be used in a method of treating cancer and/or a method of reducing the mobility of cancer cells according to the methods disclosed herein. The composition of the present invention may be a composition in any form as disclosed herein, for example a liquid or solid. In certain embodiments, the compound is an antibody, e.g., an anti-Wnt antibody, e.g., an antibody that binds to SEQ ID NO: 7 or the amino acid sequence LRAKYSLFKPPTERDL (SEQ ID NO: 80). In another embodiment, the compound is a siRNA, e.g., the nucleic acid sequence WNT3-1:5'-GGAAAAAUGCCACUGCAUC-3' (SEQ ID NO: 81), WNT3-2:5'-GGAGUGUAUUCGCAUCUAC-3' (SEQ ID NO: 82) and/ Or WNT3-3:5'-GGCUUAUCUUUGCACAUGU-3' (SEQ ID NO: 83).

Nucleic acids useful for detecting Wnt proteins (eg, primers shown in Table 1 below) are also included in the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to the methods and materials disclosed herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references cited herein are incorporated by reference as such. In the event of a dispute, the present specification, including definitions, will settle. The materials, methods, and examples are intended to illustrate, but not limit, the invention.

Other features and advantages of the invention will be apparent from the detailed description and claims set forth in detail below.

details

The invention is based, at least in part, on the fact that certain frizzled (FZD) proteins, such as FZD7 and FZD8, are associated with any cancer, such as liver cancer, and Wnt3, 8b and 11 are FZD7 ligands. Therefore, the present specification particularly provides methods of using Wnt and FZD proteins, genes, FZD-specific antibodies and probes in cancer diagnosis and treatment, and methods of screening test compounds for their ability to treat cancer. Also disclosed is a compound useful for treating cancer, such as liver cancer.

I. Nucleic Acids, Proteins, Vectors and Host Cells

The terms "Frizzled", "FZD", "Frizzled protein" and "frizzled receptor" are a group of mammalian proteins related to the Drosophila frizzled gene, and are limited to the development of tissue polarity. It means a group of proteins that do a share. The frizzled group contains 10 or more mammalian genes. Representative human frizzled receptors include frizzled 1, frizzled 2, frizzled 3, frizzled 4, frizzled 5, frizzled 6, frizzled 7, frizzled 8, frizzled 9 and frizzled 10. The frizzled receptor is associated with a dynamic model of transmembrane signaling, similar to a G-protein-coupled receptor with an amino-terminal ligand binding domain.

The terms “Wnt protein”, “Wnt ligand” and “Wnt” refer to a group of mammalian proteins related to “wingless”, the Drosophila segment polarity gene. In humans, the Wnt gene group typically encodes a 38-43 kDa cysteine floating glycoprotein [having a hydrophobic signal sequence and a conservative asparagine-linked oligosaccharide consensus sequence] [eg, Shimizu et al., Cell Growth Differ 8: 1349-1358 (1997)]. The Wnt group includes members from more than 19 mammals. Representative Wnt proteins include Wnt-1, Wnt-2, Wnt-2b (also known as Wnt-13), Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt -7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-1OA, Wnt-1OB, Wnt-11, Wnt14, Wnt15 and Wnt16.

In addition to the Wnt ligand, a group of secreted frizzled-related proteins (sFRP) was isolated. It is believed that sFRP functions as a soluble endogenous modulator in Wnt signaling by competing with a membrane-spanning frizzled receptor for secreted Wnt ligand binding. sFRP can antagonize the function of Wnt by binding proteins and blocking access to receptors involved in signal transduction on the cell surface of these proteins, or by facilitating the presentation of ligands to frizzled receptors. Can be strengthened.

The term "Wnt/FZD signaling pathway" refers to a cell initiated by an interaction between a frizzled receptor such as FZD7 and one or more of its ligands, such as a Wnt protein such as Wnt3, 8b or 11 I mean my signaling pathway. Typically, the Wnt/FZD interaction involves activating the signaling pathway by binding a Wnt protein such as Wnt3, 8b or 11 to a frizzled receptor such as FZD7. In some cases, activating the Wnt/frizzled signaling pathway will induce downstream-Wnt and/or FZD-inducible genes. A “downstream Wnt/FZD regulatory gene product” is an RNA or protein that is regulated (eg, upregulated or downregulated) as a result of signal transduction by the Wnt/FZD signaling pathway.

The present invention includes the use of any FZD and Wnt nucleic acid. For example, the present invention relates to any FZD7 and 8 nucleic acid, e.g., of the representative human and mouse FZD7 (SEQ ID NOs: 1 and 3, respectively) and FZD8 (SEQ ID NOs: 4 and 6, respectively) receptors shown in Figs. Includes the use of nucleic acids encoding amino acid sequences. As another example, the present invention provides any Wnt3, 8b and 11 nucleic acid, such as the representative human and mouse Wnt3 shown in FIGS. 8A-8E (SEQ ID NOs: 7 and 13, respectively), 8b (SEQ ID NOs: 14 and 20, respectively), And the use of a nucleic acid encoding the amino acid sequence of the protein 11 (SEQ ID NOs: 21 and 27, respectively).

The invention also includes the use of a fragment of a nucleic acid sequence encoding any of the FZD and Wnt nucleic acids, such as SEQ ID NOs: 1, 3, 4, 6, 7, 13, 14, 20, 21 or 27. Fragments of FZD or Wnt nucleic acids each encode useful fragments of one or more FZD or Wnt polypeptides (e.g., human or rodent polypeptides), e.g., binding domains (e.g., CRD domains) or other useful fragments. For example, a useful fragment of the FZD line can encode an FZD receptor fragment that retains binding activity, for example a fragment corresponding to SEQ ID NO: 3 or 5. As another example, useful fragments of Wnt nucleic acids can encode Wnt polypeptide fragments that retain binding activity, e.g., fragments corresponding to any one or more of SEQ ID NOs: 8-12, 15-19, and 22-26. .

FZD and Wnt nucleic acids disclosed herein include both RNA and DNA, including genomic DNA and synthetic (eg, chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. In the case of a single chain, the nucleic acid may be a sense chain or an antisense chain. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with modified base pairing ability or increased resistance to nucleases.

An "isolated nucleic acid" is a nucleic acid, ie, a nucleic acid that is not identical to the structure of any naturally occurring nucleic acid or has a structure that is not identical to that of any transmembrane nucleic acid consisting of three or more individual genes. Thus, the term may include, for example, (a) DNA that has some sequence of a naturally occurring genomic DNA molecule, but is not flanked by both coding sequences flanking a portion of the molecule in the genome of a naturally occurring organism; (b) a nucleic acid incorporated into a prokaryotic or eukaryotic vector or genomic DNA such that a molecule not identical to any naturally occurring vector or genomic DNA is formed; (c) separate molecules such as cDNA, genomic fragments, fragments synthesized by polymerase chain reaction (PCR), or restriction fragments; And (d) a hybrid gene, that is, a recombinant nucleotide sequence that is part of a gene encoding a fusion protein. In particular (i) DNA molecules; (ii) transfected cells; And (iii) nucleic acids present in a mixture of cell clones, eg, cell clones produced in a DNA library, eg, cDNA or genomic DNA library, are excluded from the above definition.

In some embodiments, the invention includes the use of a nucleic acid sequence that is substantially homologous to an FZD or Wnt nucleic acid. A nucleic acid sequence that is “substantially homologous” to an FZD or Wnt nucleic acid is at least 75% homologous to an FZD or Wnt nucleic acid sequence encoding any one of SEQ ID NOs: 1-27. For example, a nucleic acid sequence that is substantially homologous can be at least about 80%, 85%, 90%, 95%, 98%, or about 99% homologous to the sequence encoding SEQ ID NOs: 1-27. For comparison of nucleic acids, the length of the reference nucleic acid sequence will be at least 50 nucleotides, but may be longer, for example, at least 60 nucleotides or more.

As used herein, "% homology" of two amino acid sequences or two nucleic acid sequences is described in Karlin and Altschul (1990) Proc. Nat'l Acad. Sci USA 87:2264-2268, ie, Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877]. Such algorithms are described in Altschul et al. (1990); J. Mol. Biol. 215:403-410]. The BLAST nucleotide search method is performed with the NBLAST program [score = 100, character length = 12], and as a result, a nucleotide sequence homologous to the FZD or Wnt nucleic acid molecule used in the present invention is obtained. The BLAST protein screening method is performed with the XBLAST program [score = 50, character length = 3], resulting in an amino acid sequence homologous to the reference polypeptide. To obtain a gap-formed comparative alignment, see Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402, a gapped BLAST is used. When using BLAST and gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) are used. See www.ncbi.nlm.nih.gov.

The invention also includes the use of nucleic acids to hybridize to all or part of a nucleotide sequence encoding any of SEQ ID NOs: 1-27 under stringent hybridization conditions (as defined herein), or to a complementary sequence of such nucleic acid sequence. do. The length of the hybridization portion of the hybridizing nucleic acid is typically at least 15 nucleotides (eg, 20, 25, 30 or 50 nucleotides). The hybridization portion of the hybridization nucleic acid is at least about 75% (e.g., at least 80%, 90%, 95% or 98%) homology to a portion or all of the nucleic acid sequence encoding the FZD or Wnt polypeptide, or a complementary sequence thereof. . Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers (eg, PCR primers) or diagnostic probes. Hybridization of oligonucleotide probes to nucleic acid samples is typically carried out under stringent conditions. The stability of the nucleic acid duplex or hybrid is expressed as the melting temperature, Tm, which is the temperature at which the probe dissociates from the target DNA. This melting temperature is used to define the stringency conditions required. If the sequence is associated with the probe and turns out to be substantially identical rather than completely identical, first determine the lowest temperature, i.e., the temperature at which only homologous hybridization with a specific concentration of salt (e.g., SSC or SSPE) occurs. It is useful to establish.

Thereafter, assuming that a mismatch occurs by 1% each time the Tm temperature decreases by 1°C, the final washing temperature in the hybridization reaction also decreases accordingly. If found, the final wash temperature drops to 5°C]. In practice, the width of change in Tm can be 0.5 to 1.5°C for every 1% of a mismatch. Stringent conditions included hybridization at 68° C. in 5×SSC/5× Denhardt solution/1.0% SDS, and washing at room temperature in 0.2×SSC/0.1% SDS. Stringent conditions of hardness included washing at 3×SSC at 42°C. The parameters of salt concentration and temperature can be varied to achieve an optimal level of homology between the probe and the target nucleic acid. Additional guidance on these conditions can be readily identified in the art, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; And Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N. Y.), Unit 2.10.

A nucleic acid that hybridizes to a nucleotide sequence encoding any of SEQ ID NOs: 1-27 is considered to be an "antisense oligonucleotide".

Genetic constructs (e.g., vectors and plasmids) capable of expressing the FZD and/or Wnt nucleic acid described herein are operably linked to the transcription and/or translation sequences, such as For example, expression vectors are also included in the present invention. A DNA molecule encoding a selected nucleic acid, e.g., FZD or Wnt polypeptide, is "operable to another nucleic acid molecule, e. It is "combined". For example, the selected nucleic acid can be located adjacent to another nucleic acid molecule.

The invention also includes various engineered cells containing FZD and/or Wnt nucleic acids as described herein. For example, the present invention includes transformed host cells, that is, cells into which a nucleic acid encoding FZD and/or Wnt polypeptide has been introduced (or progenitor cells into which it has been introduced) by recombinant DNA technology. Examples of prokaryotic and eukaryotic cells include mammalian cells (eg, liver cells), fungi and bacteria (eg, Escherichia coli ) and the like. Representative engineered cells of the type included in the present invention are liver cells that overexpress the FZD7 trans gene.

Cells “overexpressing FZD” have the level of expression of certain FZD proteins, such as FZD7 and/or FZD8, ie, in non-cancer cells or non-transgenic cells each derived from the same tissue type. Cancer cells and/or transgenic cells that are about 1.5 times or more, for example about 2, 3, 4 or 5 times or more. In some embodiments, FZD expression in a cell is expressed in a non-cancer cell or non-transgenic cell of a different tissue type, or in a panel of non-cancer or non-transgenic cells of a different tissue type. Can be compared. In addition, the level of expression of one type of FZD protein (eg, FZD7) can be compared to the level of expression of another FZD protein in the same cell. Methods for measuring the level of expression of a specific gene are well known in the art. Such methods include, but are not limited to, RT-PCR, real-time PCR, and a method using an antibody against a gene product.

The use of any FZD and Wnt polypeptide is also included in the present invention. Examples of the FZD polypeptide used in the present invention include human and mouse FZD polypeptides, such as the polypeptides of SEQ ID NOs: 1 and 3, respectively, and the polypeptides of SEQ ID NOs: 4 and 6, respectively. Examples of Wnt polypeptides used in the present invention include human and mouse Wnt3, 8b and 11 polypeptides, such as the polypeptides of SEQ ID NOs: 7, 13, 14, 20, 21 and 27. Any fragments of FZD and Wnt polypeptides, such as fragments of SEQ ID NOs: 1, 3, 4, 6, 7, 13, 14, 20, 21 and 27, are also used in the present invention. Fragments of FZD and Wnt polypeptides may comprise one or more binding domains or other useful portions of full-length FZD and Wnt polypeptides. For example, useful fragments of FZD and Wnt polypeptides include, but are not limited to, fragments that retain binding activity (e.g., SEQ ID NOs: 2, 5, 8-12, 15-19 and 22-26). .

The terms “protein” and “polypeptide” are both meant to mean any chain of amino acids, regardless of length or post-translational modification (eg, glycosylation or phosphorylation). Therefore, the terms “frizzled protein”, “Wnt protein”, “frizzled polypeptide” and “Wnt polypeptide” refer not only to a full-length naturally produced isolated protein, but also a recombinant or synthetically produced polypeptide, which is a naturally occurring full-length protein Or a polypeptide corresponding to a fragment of a naturally produced or synthesized full-length polypeptide.

As described above, the terms “frizzled polypeptide” and “Wnt polypeptide” include biologically active fragments of naturally occurring or synthesized FZD and Wnt polypeptides, respectively. Protein fragments can be produced by any of a variety of methods known to the person skilled in the art, such as by proteolysis or by recombinant methods by chemical synthesis. Internal or terminal fragments of a polypeptide can be produced by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of the nucleic acid encoding the polypeptide. Expression of such mutated DNA can produce polypeptide fragments. Digestion with "end-nibbling" endonucleases can produce DNA encoding an array of fragments. The DNA encoding the protein fragment may also be produced using random shearing of oligonucleotides, restriction digestion, chemical synthesis, amplification of DNA using polymerase chain reaction, or a combination of the above methods. Fragments can also be chemically synthesized using techniques known in the art, for example using conventional Merfield solid phase FMOC or t-Boc chemical methods.

The purified or isolated compound is a composition comprising at least 60% by weight of the desired compound, for example, FZD polypeptide, Wnt polypeptide or antibody. For example, the preparation may contain 75% by weight or more (eg, 90% by weight or more, 95% by weight or more, or 99% by weight or more) of the target compound. Purity can be determined by any suitable method known in the art, such as column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

In certain embodiments, the FZD and Wnt polypeptides comprise sequences that are substantially homologous to all or part of the naturally occurring FZD and Wnt polypeptides. Polypeptides that are "substantially homologous" to the FZD and Wnt polypeptide sequences disclosed herein are at least 65% (eg, 75%, 80%, 85%, 90%, 95%) to the amino acid sequence represented by SEQ ID NOs: 1-27. % Or at least 99%, e.g., 100%) homologous (measured as described herein) amino acid sequences. For comparison purposes, the length of the reference FZD and Wnt polypeptide sequences may be at least 16 amino acids, such as at least 20 or 25 amino acids.

"Conservative amino acid substitution" means a case where an amino acid residue is substituted with an amino acid residue having a similar side chain. A group of amino acid residues with similar side chains has been defined in the art. This group comprises amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e. , Glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chain Amino acids having an aromatic side chain (eg, threonine, valine, isoleucine) and amino acids having an aromatic side chain (eg, tyrosine, phenylalanine, tryptophan, histidine).

The present invention also provides a FZD (e.g., FZD7 and/or FZD8) polypeptide or a portion of a Wnt (e.g., Wnt3, Wnt8b and/or Wnt11) polypeptide is not related thereto (e.g., a marker polypeptide or fusion Partner) to produce a fusion protein, including the use of fusion proteins (and nucleic acids encoding such fusion proteins). For example, the polypeptide is fused to a hexa-histidine tag or a FLAG tag to facilitate purification of a polypeptide expressed in bacteria, or fused to a hematoglutinin tag or a FLAG tag to purify a polypeptide expressed in eukaryotic cells. Can promote. The invention also includes the use of an isolated polypeptide (and a nucleic acid encoding the polypeptide) comprising, for example, a first portion and a second portion, wherein the first portion is, for example, FZD or Wnt A polypeptide, and the second portion comprises an unrelated polypeptide such as an immunoglobulin constant region (Fc) or detectable marker.

The fusion partner may be, for example, a polypeptide that promotes secretion, eg, a secretory sequence. The polypeptide fused in this way is usually referred to as a preprotein. The secretory sequence can be cleaved by the host cell to form a mature protein. The invention also includes nucleic acids that encode FZD and/or Wnt polypeptides fused to a polypeptide sequence to produce an inactive whole protein. The entire protein can be converted into the active form of the protein by cleaving the inactivation sequence.

II. How to detect cancer

Without being bound by any theory, a number of FZD proteins such as FZD7 and FZD8, and FZD ligands such as Wnt3 and 11 are very important in cancer such as liver cancer. In particular, it is believed that liver cells overexpress FZD7 and Wnt3 early in the transformation process, for example, before HCC progresses. Similarly, these cells often express less FZD8 and/or Wnt11. Wnt3, 8b and 11 are thought to be FZD7 ligands.

Therefore, the present invention is a method of detecting cancer cells, that is, a method of facilitating diagnosis of the presence and severity of cancer in a patient (e.g., tumor grade and tumor burden, etc.), Methods of facilitating measurements and methods of assessing a patient's responsiveness to therapy (e.g., methods of measuring therapeutic effect by assessing tumor loading during or after chemotherapy practice) are provided.

Detection is the detection of polynucleotides (e.g., FZD7, FZD8, Wnt3 and/or Wnt11 polynucleotides) differentially expressed in cancer cells (e.g., when compared to non-cancer cells) and/or It can be based on the detection of polypeptides (eg, FZD7, FZD8, Wnt3 and/or Wnt11 polypeptides) encoded by polynucleotides that are differentially expressed in cancer cells. The detection method of the present invention can be carried out in vitro or in vivo, on a biological sample, for example, isolated cells and/or whole tissues.

As used herein, "biological sample" refers to a biological tissue or fluid sample containing a nucleic acid or polypeptide such as an FZD7 protein, polynucleotide or transcript. Such samples include, but are not limited to, tissues obtained from the liver, lungs, lymph nodes, colon, stomach, pancreas, bile ducts, small intestine and/or esophagus. Biological samples also include tissue specimens such as biopsy and autopsy samples, frozen specimens taken for anatomical use, blood, plasma, serum, sputum, feces, tears, mucus, bile, saliva, lymph, hair, skin, etc. May be. Biological samples may also include explants derived from patient tissues, primary and/or modified cell culture fluids. Biological samples are typically eukaryotic organisms such as primates such as chimpanzees or humans; small; Words; Goat; amount; dog; cat; Rodents such as guinea pigs, rats or mice; rabbit; bird; reptile; Or obtained from fish. Typically, a sample is provided by isolating a sample of cells from an animal, but providing previously isolated cells (e.g., cells isolated from others at different times and/or cells isolated for various purposes), or in vivo May be provided by performing the method of the present invention Tissues that have undergone certain treatments or have been stored for a very long time may be used.

In some embodiments, methods of detecting cancer cells are provided by detecting that transcripts that are differentially expressed in cancer cells (e.g., FZD7, FZD8, Wnt3 and/or Wnt11 transcripts) are expressed in the cell. Any of a number of known methods includes, for example, hybridizing mRNA with an appropriate hybridization probe to detect transcripts; A method of detecting a transcript by polymerase chain reaction using a specific oligonucleotide primer; And a method of hybridizing in vitro using an appropriate hybridization probe. This method can be used to detect and/or measure the mRNA levels of genes that are differentially expressed in cancer cells. In some embodiments, the method comprises: a) contacting a sample with a polynucleotide corresponding to a differentially expressed gene as described herein under conditions capable of hybridization; And b) if hybridized, detecting the hybridized material.

When compared with an appropriate control, the detection result of differential hybridization becomes an index indicating the presence of a polynucleotide sample that is differentially expressed in cancer cells. For example, suitable controls include samples that are not cancer cells, samples known to not contain polynucleotides that are differentially expressed in cancer cells, and have the same "sense" sequence as polynucleotides that are differentially expressed in cancer cells. Labeled polynucleotides are used. Conditions for hybridization are known in the art, and are described in more detail above. Detection may also be performed by any known method, such as in vitro hybridization, PCR (polymerase chain reaction) and/or RT-PCR (reverse transcription-PCR), or by using a combination of known techniques. Many labeling and labeling methods for polynucleotides are known in the art, and these methods can be used in the assay methods of the present invention. Hybridization specificity can be determined by comparison with an appropriate control.

In general, polynucleotides comprising at least 10 nt, at least 12 nt, or at least 15 nt of contiguous nucleotides of the polynucleotides disclosed herein, e.g., polynucleotides having the sequences described herein, have a variety of uses, e. It can be used as a probe or PCR primer for measuring and / or detecting the transcription level of the polynucleotide differentially expressed in. As will be appreciated by one of skill in the art, the probe can be labeled to be detectable and contacted with an array comprising immobilized polynucleotides obtained, for example, from a test sample (eg, mRNA). Alternatively, probes can be immobilized on the array and on test specimens labeled to be detectable. The use of the above and other variations of the method of the present invention are well known to those skilled in the art and are within the scope of the present invention.

Nucleotide probes can be used to detect the expression of a gene corresponding to a given polynucleotide. In Northern blot, the mRNA is separated by electrophoresis and brought into contact with the probe. Probes are detected when hybridized with an mRNA species of a specific size. The amount of hybridization can be quantified to determine the relative amount of expression. The probe can be used for in situ hybridization to cells for which expression is to be detected. Probes can also be used for in vivo diagnostic detection of hybridization sequences. Probes can be labeled with radioactive isotopes or other types of detectable labels such as chromophores, fluorophores and/or enzymes. Other examples of nucleotide hybridization assays are described in WO 92/02526 and US Pat. No. 5,124,246.

PCR is another means for detecting small amounts of target nucleic acid [eg, Mullis et al., Meth. Enzymol. (1987) 155:335; US Patent No. 4,683,195; And 4,683,202]. Two primer oligonucleotides that hybridize to the target nucleic acid can be used to prime the reaction. The primers may be comprised within the polynucleotides described herein, or may consist of sequences contained in the 3'→5' direction to this polypeptide. After amplifying the target by standard PCR method, the amplified target nucleic acid can be detected by a method known in the art, for example, Southern blot. mRNA or cDNA is also a conventional blotting technique as disclosed in Sambrook et al., “Molecular Cloning: A Laboratory Manual” (New York, Cold Spring Harbor Laboratory, 1989) (eg, without PCR amplification process). It can also be detected by (for example, Southern blot, Northern blot, etc.). In general, cDNA or mRNA produced from mRNA using a polymerase can be purified and separated by gel electrophoresis and transferred to a solid support such as nitrocellulose. This solid support is washed after exposure to the labeled probe, and any probes that have not hybridized can be removed. The duplex containing the labeled probe can then be detected.

The method using the PCR amplification method can be performed on DNA derived from one or more cells. The use of the polymerase chain reaction is described in the literature [Saiki et al. (1985) Science 239:487], and the study on the technique is described in the literature [Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory, 1989; pp.14.2-14.33). The detectable label can be included in the amplification reaction. Suitable detectable labels include fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), rhodamine, Texas red, phycoerythrin, allophicocyanine, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA)), Radiolabels (eg, ³² P, ³⁵ S, ³ H, etc.), and the like. The label may be a two stage system, that is, the polynucleotide is conjugated to a high affinity binding partner such as avidin, biotin, hapten, etc. bound to a specific antibody, and the binding partner is detected. It is conjugated to a possible label. The label can be conjugated to one or both of the primers. Alternatively, the nucleotide pool used in the amplification process is labeled to incorporate the label into the amplification product.

In one embodiment, the expression level is assessed by real-time PCR. RNA is isolated from the sample of interest. PCR primers are designed to amplify a specific gene of interest. The accumulation of PCR products is determined using a double-labeled fluorescent oligonucleotide probe. The probes are labeled with two different fluorescent dyes, a 5'end reporter dye and a 3'end quenching dye. The oligonucleotide probe is selected to be homologous to the internal target sequence present in the PCR amplicon. When this probe is in its original state, energy transfer occurs between the two fluorophores, and the fluorescence emission phenomenon stops. During the amplification process of PCR, the probe is cleaved by the 5'nuclease activity of Taq polymerase. Therefore, the reporter is no longer present in rapid contact with the quenching material, and at this time, it can be seen that the luminous intensity increases. A representative method of detecting FZD expression using real-time PCR is provided in the Examples section below. Primers can also be used in other methods such as RT-PCR. These assays provide a means of quantitative measurement of nucleic acids.

In another embodiment, a method of detecting cancer cells by detecting the expression of proteins that are differentially expressed by the cells (eg, FZD7, FZD8, Wnt3 and/or Wnt11 proteins) is provided. Any of a number of known methods can be used as the detection method, for example, using a binding compound such as an antibody or antigen binding fragment thereof (a fragment useful in ELISA and/or Western blot methods). Such antibodies may be polyclonal or monoclonal antibodies, and may also be labeled with detectable markers (eg, fluorophores, chromophores or isotopes, etc.). If appropriate, this compound can be attached to a solid support such as beads, plates, filters, resins, and the like. A method of detecting whether a compound/target complex has been formed can be carried out by contacting this complex with an additional compound (e.g., a secondary antibody), that is, a compound that specifically binds to the first compound (or complex). have. As with the first compound, additional compounds may be attached to a solid support and/or may be labeled with a detectable marker.

Materials necessary to perform the detection methods described herein can be provided as part of a kit. Therefore, the present invention further provides detection of whether and/or the level of polynucleotide differentially expressed in cancer cells and/or a polypeptide encoded by it is present in a biological sample (e.g., differentially expressed genes of interest It also provides a detection kit for detecting by detecting the mRNA encoded by the method. Methods of using this kit can be performed by clinical laboratories, laboratory laboratories, medical practitioners or individuals. The kit of the present invention, that is, a kit for detecting a polypeptide encoded by a polynucleotide differentially expressed in cancer cells, may include a portion that specifically binds to the polypeptide, for example, an antibody. The kit of the present invention used to detect polynucleotides that are differentially expressed in cancer cells may include a moiety that hybridizes specifically to such polynucleotides. The kit may also provide additional components useful in the detection method, such as buffers, developing reagents, labels, reaction surfaces, detection means, control samples, standards, instructions and interpretive information.

The invention also relates to a method for detecting/diagnosing neoplasms or pre-neoplastic conditions in mammals (eg humans). As used herein, the term "diagnosis" generally refers to measuring a patient's sensitivity to a disease or disorder, measuring whether an individual has a current disease or disease, or determining the prognosis of an individual with a disease or disorder. Observation (e.g., observing the pre- or metastatic cancer state, the stage of cancer, or the responsiveness of the cancer to therapy), and differential diagnosis (e.g., by observing the condition of the individual, the effectiveness or efficacy of the therapy) To provide information about).

One exemplary detection/diagnosis method includes the following steps: (a) obtaining a biological sample (eg, liver tissue) from a mammal (eg, human); (b) detecting the presence or absence of FZD7, FZD8, Wnt3 and/or Wnt11 gene products (eg, protein or mRNA) in the sample; And (c) comparing the amount of the FZD7, FZD8, Wnt3 and/or Wnt11 gene product with the amount of the FZD7, FZD8, Wnt3 and/or Wnt11 gene product in the control sample. According to this method, an increase in the level of the FZD7 and/or Wnt3 gene product in the sample and/or a decrease in the level of the FZD8 and/or Wnt11 gene product in the sample means that the individual has cancer or a precancerous condition such as liver cancer or liver cancer It indicates that there is a risk of development into the market.

Ascertaining increased FZD7 and/or Wnt3 protein levels and/or decreased FZD8 and/or Wnt11 protein levels in accordance with the present invention can identify patients who are likely to benefit from a particular treatment. For example, a biological sample obtained from an individual after performing the first treatment (e.g., an individual undergoing surgery) has an elevated level of FZD7 and/or Wnt3 protein and/or a decreased level of FZD8 and/or Wnt11 protein. It is screened for whether or not, and it can be confirmed that this protein level is an indicator of whether the tumor tissue remains. Similarly, the tissue surrounding the cut site of the tumor that was surgically removed (e.g., tissue peri-tumor) has an elevated level of FZD7 and/or Wnt3 (relative to the surrounding tissue) or FZD8 and/or It can be observed whether the level of Wnt11 has decreased (e.g., by immunophotometry), and these observations indicate whether there is a possibility of disease progression in this tissue or whether the tumor has been incompletely removed. Can be. This ability to identify patients allows the treatment to be tailored to specific patient needs. Subjects undergoing treatment other than surgery, e.g. chemotherapy or radiation therapy, may also be observed, with increased levels of FZD7 and/or Wnt3 or decreased levels of FZD8 and/or Wnt11 in samples obtained from such subjects. That is, it becomes an indicator of whether continuous treatment is needed. One of skill in the art will also appreciate that the method disclosed herein can be used to determine the stage of cancer (eg, liver cancer) to optimize the treatment method.

III. How to identify compounds that can cure cancer

The present invention provides a method of screening for whether a test compound has the ability to treat cancer, such as liver cancer. As described herein, "test compound" refers to any compound that can be screened using the methods described herein. For example, the test compound can be, for example, a small organic molecule or a small inorganic molecule (molecular weight = less than 1000 Da). Alternatively or additionally, the test compound may be a polypeptide (eg, a polypeptide having a random or predetermined amino acid sequence or a naturally occurring or synthetic polypeptide) or a nucleic acid such as a DNA or RNA molecule. Test compounds may be naturally occurring (eg, vegetable or natural products), or may be synthetic, or may contain both natural and synthetic ingredients. The formal weight of the test compound may be less than about 10,000 g/mole, less than 5,000 g/mole, 1,000 g/mole, or less than about 500 g/mole. For example, the test compound may be any organic or inorganic compound (e.g., heteroorganic compound or organometallic compound), amino acid, amino acid analog, polypeptide, peptidomimetic (e.g., peptoid ), oligopeptides (e.g., about 5 to about 25 amino acids in length, preferably about 10 to 20 amino acids, or 12 to 18 amino acids, preferably 12, 15 or 18 amino acids), nucleotides, nucleotide analogues, poly Nucleotides, polynucleotide analogs, ribonucleic acids, deoxyribonucleic acids, antisense oligonucleotides, ribozymes, sugars, lipids (e.g., sphingolipids) and/or fatty acids or any combination of these substances.

The term “antagonist” or “inhibitor” of Wnt/FZD signaling (eg, Wnt/FZD7 signaling) is, for example, a Wnt protein (eg, Wnt3, 8 and/or 11) and/or Binds to the FZD receptor (e.g., FZD7) and/or as measured in known assays for Wnt/FZD signaling [e.g., β-catenin levels, Tcf and Lef transcription factors or other downstream Wnt /Measurement of expression of oncogenes controlled by frizzled regulatory gene products], partially or entirely, means a compound that blocks or inhibits Wnt/FZD signaling (eg, Wnt/FZD7 signaling). Examples of inhibitors include antibodies directed against Wnt or FZD proteins (described in the Examples section below for examples of anti-Wnt3 antibodies), modified forms of Wnt or FZD proteins, naturally occurring and synthetic ligands. , Antagonists, agonists, antibodies, small chemical molecules, and the like. Assays for detecting inhibitors or antagonists are described in more detail below.

Library of test compounds

In certain embodiments, the screening method of the present invention utilizes a library of test compounds. "Library" is a collection of compounds synthesized by various combinations of one or more starting components (eg, individual compounds in the form of mixtures or physically separated). At least some of the compounds must be different from at least some of the other compounds present in the library. Libraries can be, for example, 5, 10, 50, 100, 1000, or 10,000, 50,000 or 100,000 or more different compounds (i.e., some compounds in the library may exist in duplicate or more than one, but simply the same But not multiple copies of the compound). Each of the different compounds will be present in an amount that can be confirmed by some means, for example, in an amount that can be separated, analyzed and/or detected using a receptor or suitable probe. The actual amount of this compound required to ascertain the presence of each different compound will vary depending on the method actually used and may also vary as separation, detection and analysis techniques advance. When a compound is present in the mixture in an actual equivalent molar amount, for example, each compound can be detected in an amount equivalent to 100 pmoles. The library is a library of each compound (e.g., a library prepared through parallel synthesis or a pool and split pool method, and substantially exists as a compound in a single form per well) and each target compound A mixture containing substantially the same molar amount (ie, a mixture in which a single compound is hardly present) may be included. Any one of the libraries makes it possible to identify the active compounds found in the assay.

Test compounds can be screened individually or in a parallel manner. An example of parallel screening is high-throughput drug screening for a large library of chemicals. Such libraries of candidate compounds can be prepared or purchased, for example, from Cambridge Corp., San Diego, CA. Alternatively, a group or category of compounds with enhanced efficacy can be suggested, based on previous experimentation and evidence from real life. Libraries can be designed and synthesized to contain these groups of chemicals.

Methods for synthesizing combinatorial libraries are well known in the art, and in this regard, see, for example, E.M. Gordon et al, J. Med. Chem. (1994) 37:1385-1401; DeWitt, S. H.; Czarnik, A. W. Acc. Chem. Res. (1996) 29:114; Armstrong, R. W.; Combs, A. P.; Tempest, P. A.; Brown, S. D.; Keating, T. A. Acc. Chem. Res. (1996) 29:123; Ellman, J. A. Acc. Chem. Res. (1996) 29:132; Gordon, E. M.; Gallop, M. A.; Patel, D. V. Acc. Chem. Res. (1996) 29:144; Lowe, G. Chem. Soc. Rev. (1995) 309, Blondelle et al. Trends Anal. Chem. (1995) 14:83; Chen et al. J. Am. Chem. Soc. (1994) 116:2661; U.S. Patent Nos. 5,359,115, 5,362,899 and 5,288,514; PCT Publication Nos. WO 92/10092, WO 93/09668, WO 91/07087, WO 93/20242, WO 94/08051.

Compound libraries can be prepared by a variety of methods, some of which are known in the art. For example, the "split-pool" technique can be implemented in the following manner, i.e., by placing functionalized polymer support beads into a number of reaction vessels, wherein a number of polymer supports suitable for solid-phase peptide synthesis methods are Known and some are commercially available (see, eg, M. Bodansky "Principles of Peptide Synthesis", 2nd edition, Springer-Verlag, Berlin (1993)). Different activated amino acids are added to each bead aliquot, and the reaction proceeds to obtain a plurality of immobilized amino acids (one in each reaction vessel). The derivatized bead aliquots are then washed, "pooled" (ie recombined), and the bead pool is divided again, with each aliquot placed in a separate reaction vessel. Then, another activated amino acid is added to each bead aliquot. The synthesis cycle is repeated until the length of the desired peptide is obtained. Amino acid residues added to each synthesis cycle can be randomly selected; Alternatively, amino acids are selected to be “biased” libraries such as known peptides, such as anti-genotypes, that any portion of the inhibitor can be non-randomly selected to interact with, for example, antibodies. Libraries can be provided that will provide inhibitors of known structural similarity or homology to the antibody antigen binding site. It will be appreciated that a variety of peptides, peptido mimetics or non-specific compounds can be readily prepared in this manner.

The “split-pull” technique can produce a peptide library, eg, a modulator library, that can be used to prepare a library of test compounds of the present invention. In another exemplary synthesis method, the "various library" is prepared by the method described by Hobbs DeWitt et al (Proc. Natl. Acad. Sci. USA 90:6909 (1993). For example, Hutton's "tea-bag" technique (eg Houghten et al., Nature 354:84-86 (1991)) can also be used to synthesize a library of compounds according to the invention.

Compound libraries can be screened to determine whether any member of the library possesses the desired activity, and if so, the active species can be identified. A method for screening combinatorial libraries has been disclosed [eg, Gordon et al., J Med. Chem. (same as above)]. The soluble compound library can be screened by affinity chromatography using an appropriate receptor to separate the ligand for the receptor, and then identify the isolated ligand by conventional techniques (e.g., mass spectrometry, NMR, etc.). I can. Immobilized compounds can be screened by contacting this compound with a soluble receptor, wherein the soluble receptor is preferably a detection label that may indicate that it is bound to a ligand (e.g., a fluorophore, a chromogenic enzyme, a radioactive isotope). , Light-emitting compounds, etc.). Alternatively, the immobilized compound is selectively released and diffuses through the membrane, allowing it to interact with the receptor. Exemplary assays useful for screening test compound libraries are described above.

Screening method

The present invention provides a method of identifying a compound capable of treating cancer, such as liver cancer. Although the applicants of the present invention are not wishing to be limited to any particular theory of biological mechanisms related thereto, such compounds are particularly suitable for (1) Wnt/FZD signaling (e.g. It is believed to modulate the binding of and/or signal transduction by reduction (eg, prevention) of Wnt/FZD-mediated transcription] and/or (2) expression of FZD7, FZD8, Wnt3 and/or Wnt11.

In any aspect of the invention, the method of screening for such compounds comprises: (i) binding to the FZD7, Wnt3, Wnt8b and/or Wnt11 polypeptide among the group of test compounds, and FZD7 and its ligands (e.g., Wnt3, Wnt8b and / Or Wnt11) to modulate (e.g., increase or decrease) the interaction between) and/or the transcription and/or translation of FZD7, FZD8, Wnt3, Wnt8b and/or Wnt11 (e.g., increase or decrease) Identifying the compound to be obtained; And optionally, (ii) modulating Wnt/FZD signaling, reducing cancer cell mobility, reducing the accumulation of β-catenin in cancer cells, and/or treating cancer in vitro or in vivo. It is carried out by further testing the ability of the compound. Test compounds that bind to FZD7, Wnt3, Wnt8b and/or Wnt11 polypeptide modulate the interaction between FZD7 and its ligands (e.g., Wnt3, Wnt8b and/or Wnt11), or FZD7, FZD8, Wnt3, Wnt8b And/or the transcription and/or translation of Wnt11, and thus is referred to herein as a “candidate anticancer agent”. Candidate anticancer agents are also further tested, as a result of which they can modulate Wnt/FZD signaling in vitro or in vivo, can reduce the mobility of cancer cells, and reduce/reduce the accumulation of β-catenin in cancer cells. It is considered to be an "anti-cancer agent" herein as it is believed to be capable of causing or treating cancer. In the screening method of the present invention, a candidate anticancer agent may be tested to determine whether it is an anticancer agent, but it does not have to be. The assays of the present invention can be performed in biological samples, whole cell preparations, and/or extracellular cell-free systems.

In one aspect, the invention encompasses methods of screening test compounds to identify compounds that bind to FZD polypeptides, e.g., FZD7 polypeptides, and/or Wnt polypeptides, e.g., Wnt3, Wnt8b and/or Wnt11 polypeptides. do. Whether the test compound binds to the FZD or Wnt polypeptide is determined by reversibly or irreversibly immobilizing the test compound(s) or Wnt or FZD polypeptide in vitro on the well surface of a substrate, e.g., a 96-well polystyrene microtiter plate. , Can be detected. Methods for immobilizing polypeptides and other small molecules are well known in the art. For example, in a microtiter plate, a polypeptide in solution (typically, a concentration of 0.05 to 1 mg/ml in a volume of 1 to 100 μl) is added to each well, and the plate is placed at room temperature for a predetermined time, e.g., , It can be coated with FZD or Wnt polypeptide by incubating at room temperature to 37° C. for 0.1 to 36 hours. Polypeptides that do not bind to this plate can be removed by adding excess solution to the plate and shaking, and then washing the plate with water or buffer (once or repeatedly). Typically the polypeptide is in water or in a buffer. Thereafter, the plate is washed with a buffer solution to remove the bound polypeptide. To block the free protein-binding site on the plate, the plate can be blocked with a protein unrelated to the bound polypeptide. For example, 300 µl of bovine serum albuminate (BSA) at a concentration of 2 mg/ml in Tris-HCl may be used. Suitable substrates include substrates containing limited crosslinking chemicals (e.g., plastic substrates such as polystyrene, styrene or polypropylene substrates (e.g., from Corning Costa Corporation, Miami Cambridge)). If desired, particles such as beaded agarose or beaded sepharose may also be used as the substrate. The test compound is then added to the coated plate to allow the FZD or Wnt polypeptide to bind (37° C., 0.5-12 hours). Thereafter, the flat plate can be rinsed as described above. The skilled person will understand that this method can be implemented in many variations. For example, the method may include coating a substrate with a test compound and adding a Wnt or FZD polypeptide to the substrate-binding compound.

It is known in the art whether FZD or Wnt is bound to a second compound, e.g., a test compound or binding partner as described above (e.g., Wnt3, 8b and/or 11 for FZD7; described in more detail below). It can be detected by any of the various methods described. For example, antibodies that specifically bind to FZD or Wnt polypeptides (i.e., anti-FZD antibodies or anti-Wnt antibodies, e.g., polyclonal anti-Wnt3 antibodies described in the Examples section below) can be used in immunoassays. have. If desired, the antibody can be labeled (eg, fluorescently labeled or labeled with a radioactive isotope) and detected directly (eg, West and McMahon, J. Cell Biol. 74:264, 1977]. Alternatively, a second antibody (eg, an anti-FZD antibody or a labeled antibody that binds to the Fc portion of an anti-Wnt antibody) can be used for detection. In an alternative method of detection, the FZD or Wnt polypeptide is labeled (eg, with a radioactive isotope, fluorophore, chromophore, etc.), and this label is detected. In another method, the FZD or Wnt polypeptide is produced as a fusion protein with a protein that can be optically detected using, for example, green fluorescent protein (detectable under UV light). In an alternative method, the polypeptide is prepared as a fusion protein with an enzyme that retains detectable enzymatic activity, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase or glucose oxidase. Genes encoding all of the above enzymes can be easily used by being cloned by a person skilled in the art. If desired, the fusion protein may contain an antigen or epitope that can be detected and measured with polyclonal or monoclonal antibodies using conventional methods. Suitable antigens include enzymes (e.g. horseradish peroxidase, alkaline phosphatase and β-galactosidase) and non-enzymatic polypeptides (e.g., serum proteins such as BSA and globulin, and milk proteins such as For example, casein).

In various methods for identifying a polypeptide (e.g., a test polypeptide) that binds to an FZD or Wnt polypeptide, a conventional two-hybrid assay for protein/protein interaction can be used (e.g. For example, Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578, 1991; Fields et al., US Patent No. 5,283,173; Fields and Song, Nature, 340:245, 1989; Le Douarin et al., Nucleic Acids Research, 23:876, 1995; Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10315-10320, 1996; And White, Proc. Natl. Acad. Sci. USA, 93:10001-10003, 1996]. In general, the two-hybrid method involves reconstituting two separate domains of a transcription factor. One fusion protein comprises an FZD or Wnt polypeptide fused to either a transactivator domain or a DNA binding domain of a transcription factor (eg, Gal4). The other fusion protein contains the binding partner of the polypeptide contained in the test polypeptide or the first fusion protein [which is fused to either the transactivator domain of the transcription factor or the DNA binding domain]. The transcription factor is reconstituted by binding of the FZD or Wnt polypeptide to the test compound or binding partner. Whether the transcription factor is reconstituted can be confirmed by detecting the expression of a gene (ie, a reporter gene) operably bound to a DNA sequence bound by the DNA binding domain of the transcription factor. Kits for implementing various two-hybrid methods are commercially available (eg, Clontech, Palo Alto, Calif.).

In another aspect, the invention includes methods of screening test compounds to identify compounds that modulate protein-protein interactions between FZD and Wnt polypeptides. For a method useful for screening at high throughput for compounds capable of modulating protein-protein interactions between transcriptional regulatory factors, see Leporcelet et al, Cancer Cell 5: 91-102 (2004); Herein incorporated by reference as such. Typically the first compound is provided. The first compound is an FZD (eg, FZD7) or Wnt (eg, Wnt3, 8b or 11) polypeptide or a biologically active fragment thereof. The second compound is labeled differently from the first compound. The second compound is an FZD (eg, FZD7) or Wnt (eg, Wnt3, 8b or 11) polypeptide or a biologically active fragment thereof. Test compounds are provided. The first compound, the second compound and the test compound are brought into contact with each other. Then, the amount of the label bound to the first compound is measured. Changes in the protein-protein interaction between the first compound and the second compound, as assessed by the binding of the label, indicate that this test compound is useful in modulating the protein-protein interaction between the FZD and the Wnt polypeptide. It is an indicator.

In certain embodiments, the provided first compound is attached on a solid support. The solid support includes, for example, a resin (eg, agarose and beads) and a multi-well plate. In certain embodiments, the method includes a washing step after the contacting step, whereby bound and unbound labels are separated.

In certain embodiments, a plurality of test compounds are contacted with the first compound and the second compound. Different test compounds can be contacted with other compounds at once or individually. In certain embodiments, each of the test compounds is contacted with a first compound and a second compound within a respective well. For example, the method can screen a library of test compounds. The library of test compounds has already been described above. Libraries may contain, for example, natural products, organic chemicals, peptides and/or modified peptides such as D-amino acids, unusual amino acids and N-substituted amino acids. Typically, the library is in a format suitable for screening in a multi-well plate (eg, a 96-well plate). An assay that is automated in a multi-well manner [many of the steps included in this assay are controlled by a computer and executed by an automated device] is particularly useful. The library can also be used in other ways, for example as a synthetic chemical library that is immobilized on a solid support, which can also be easily released in microdroplets.

In certain embodiments, the first compound is an FZD7 polypeptide or fragment thereof, and the second compound is a Wnt polypeptide, eg, Wnt3, 8b or 11 polypeptide or fragment thereof. In another embodiment, the first compound is a Wnt polypeptide such as Wnt3, 8b or 11, or a fragment thereof, and the second compound is an FZD7 polypeptide or fragment thereof. The solid support to which the first compound is bound may be, for example, Sepharose beads, SPA beads (microspheres containing a light emitting agent), or a multi-well plate. SPA beads can be used, for example, in a scintillant proximity assay when the assay is performed omitting the washing step. Sepharose beads can be used when the assay is performed without omitting the washing step. The second compound may be labeled with any detectable label, such as a radioactive label, a fluorescent agent, biotin, a peptide tag, or an enzyme fragment. The second compound may also be radiolabeled with, for example, ¹²⁵ I or ^{3 H.}

In certain embodiments, the enzymatic activity of the enzyme expressed as a fusion protein carrying the first compound or the second compound, or chemically conjugated to the first compound or the second compound, is used to detect the bound protein. Is used. Also included are binding assays that detect binding proteins using standard immunological methods. In certain other embodiments, the interaction of the Wnt and FZD polypeptides or fragments thereof is such that there is an appropriate overlap between the donor emission spectrum and the acceptor excitation spectrum, such that the fluorophore is proximate through the protein-protein interaction of the FZD and Wnt polypeptides. When the non-radioactive energy is efficiently transferred when present, a donor fluorophore covalently bound to an FZD or Wnt polypeptide [e.g., a fluorescent group chemically conjugated to FZD or Wnt, or an FZD or Wnt-GFP chimeric protein It is detected by fluorescence resonance energy transfer (FRET) between the green fluorescent protein (GFP) variant, which is expressed as, and a receptor fluorophore covalently bonded to the substrate protein.

In another embodiment, protein-protein interactions are detected by reconstitution of domains of an enzyme, eg, beta-galactosidase [Rossi et al, Proc. Natl. Acad. Sci. USA 94:8405-8410 (1997)].

In another embodiment, the protein-protein interaction images the fluorescence ratio of the appropriate chimeric construct of the FZD and Wnt polypeptides in the cell [Bacskai et al, Science 260:222-226 (1993)], or a two-hybrid Variations of the assay [Fearon et al, Proc Natl Acad Sci USA 89:7958-7962 (1992); Takacs et al, Proc Natl Acad Sci USA 90:10375-10379 (1993); Vidal et al, Proc Natl Acad Sci USA 93:10321-10326 (1996)] That is, it was evaluated by carrying out a method using the appropriate constructs of FZD and Wnt polypeptides, and was also carried out in accordance with a high-throughput assay, so that the FZD/Wnt interactions A compound that inhibits the action is detected. This embodiment has the advantage of ensuring the cell permeability of the test compound.

For example, in one assay, the FZD or Wnt polypeptide or fragment thereof is adsorbed on an ELISA plate. The FZD or Wnt polypeptide is then exposed to the test compound → glutathione-S-transferase (GST)-binding partner fusion protein, such as GST-FZD or GST-Wnt polypeptide fusion protein. Bound protein is detected as goat anti-GST antibody, alkaline phosphatase (AP)-coupling anti-goat IgG and AP substrate. Compounds that interfere with protein-protein interactions reduce the AP signal in experiments on ELISA plates.

In another aspect, the invention provides a method for the identification of test compounds that modulate (eg, increase or decrease) the expression of FZD and/or Wnt polypeptides. The method includes contacting the FZD and/or Wnt nucleic acid with a test compound and then measuring the expression of the encoded FZD and/or Wnt polypeptide. In a related aspect, the invention features a method of identifying a compound that modulates (e.g., increases or decreases) the expression of an FZD and/or Wnt polypeptide, the method comprising: (a) an FZD and/or Wnt nucleic acid A cell line integrated into a preparative construct comprising a sequence or fragment thereof or an allelic variant thereof; Or (b) measuring the expression of the FZD polypeptide in the presence of a test compound, present in a cell population or cell line that selectively expresses FZD and/or Wnt in its natural state, or after adding this test compound. After measuring the expression, it is carried out by measuring the expression of FZD and/or Wnt protein.

Once the FZD and Wnt nucleic acids disclosed herein have been identified, these nucleic acids can be cloned into various host cells (e.g., mammalian cells, insect cells, bacteria or fungi) to perform such assays in whole cells.

In certain embodiments, the isolated nucleic acid molecule encoding the FZD and/or Wnt polypeptide modulates the expression of FZD and/or Wnt in vivo (e.g., in FZD and/or Wnt-producing cells) (e.g. For example, it is used to identify compounds that increase or decrease). In this embodiment, cells expressing FZD (e.g., FZD7 and/or FZD8) and/or Wnt (e.g., Wnt3, Wnt8b or Wnt11) are cultured, and the test compound (or mixture of test compounds) Following exposure to, the level of FZD and/or Wnt expression is compared to the level or activity of FZD and/or Wnt expression in cells (or cells identical to previous cells, but not exposed to the test compound(s)). Standard quantitative assays of gene expression can be used.

Expression of FZD and Wnt can be measured by methods known in the art, for example, Northern blot PCR assay or RNAse protection assay using the nucleic acid molecule of the present invention as a probe. Other examples include enzyme-linked immunosorbent assay (ELISA), radioactive immunoassay (RIA), and fluorescence activated cell sorting (FACS). The level of expression in the presence of the test molecule, when compared to the level of expression in the absence of the test molecule, will provide an indicator of whether it is a test compound that modulates the expression of the FZD and/or Wnt polypeptide.

In another aspect, the present invention provides a method for screening a test compound using a cell system susceptible to disruption occurring in one or several transcription/translation components.

In certain embodiments, the method comprises identifying candidate compounds that step down the accuracy of FZD and/or Wnt translation, e.g., maintain an appropriate reading framework during translation and terminate translation at a stop codon. . The method involves constructing a cell that produces a reporter polypeptide that is detectable only if it remains in one reading framework or if the normal process of terminating translation at the stop codon is entangled. The method further includes the step of contacting the cell with a test compound to observe whether the production of the reporter polypeptide is increased or decreased.

In another embodiment, the cell line is a cell-free extract, the method comprising measuring transcription or translation in vitro. Conditions are selected to increase or decrease the transcription or translation of the reporter by adding a transcription or translation modifier to the cell extract.

One way to identify candidate compounds depends on the transcription-reactive gene product. The method includes constructing cells in which the amount of reporter molecule production changes (i.e., increases or decreases) under conditions in which the amount of cellular transcription of the FZD and/or Wnt nucleic acid changes (i.e., increases or decreases). . Specifically, this reporter molecule is encoded by a nucleic acid that is transcriptionally bound to a sequence constructed and aligned to induce a relative change in the production amount of the reporter molecule as the amount of transcription of the FZD and/or Wnt nucleic acid changes. The gene sequence encoding the reporter can be fused to, for example, some or all of the gene encoding the transcription-reactive gene product, and/or some or all of the genetic elements that control the production of the gene product. Alternatively, the transcription-reactive gene product can directly or indirectly promote the transcription of a gene encoding a reporter. The method further includes contacting the cell with a test compound and determining whether the test compound increases or decreases the production of reporter molecules in the cell.

Alternatively, the method of identifying candidate compounds may vary depending on the translation-reactive gene product. The method includes constructing a cell with a changed (ie, increased or decreased) amount of intracellular translation of the FZD and/or Wnt nucleic acid. Specifically, the reporter molecule is encoded by a nucleic acid that is translatably linked to a sequence constructed and aligned to relatively increase or decrease the amount of production of the reporter molecule when the amount of transcription of the FZD and/or Wnt nucleic acid changes. The gene sequence encoding the reporter can be fused to, for example, some or all of the gene encoding the transcription-reactive gene product, and/or some or all of the genetic elements that control the production of the gene product. Alternatively, the transcription-reactive gene product can directly or indirectly promote the transcription of a gene encoding a reporter. The method further includes contacting the cell with a test compound and determining whether the test compound increases or decreases the production of reporter molecules in the cell.

In the above method and any of the methods described herein, a variety of reporters may be used, wherein a conventional reporter provides a conveniently detectable (eg, detected by a spectrometer) signal. For example, a reporter gene can encode an enzyme that catalyzes a reaction that alters absorbance.

Examples of the reporter molecule include, for example, β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase, exo-glucanase, glucoamylase, and radiolabeling. Included reporters. For example, whether or not a reporter molecule is produced may be determined by the enzymatic activity of a reporter gene product, for example, β-galactosidase.

Any of the methods described herein can be performed for high-throughput screening of multiple test compounds and used to identify candidate anticancer agents. High-throughput screening refers to a method capable of screening a large number of candidate compounds relatively easily and quickly.

When the test compound is identified as a candidate anticancer agent, the compound is further tested in vivo or in vitro through techniques known in the art, and if desired, it is possible to confirm whether the compound is an anticancer agent. That is, the compound is Wnt/ Whether it is possible to modulate FZD signaling, modulate the mobility of cancer cells and/or, in vitro (e.g., using isolated cells or cell-free systems) or in vivo (e.g., Animal e.g. using rodent model system) can determine whether expression is modulated

Methods for in vitro testing of candidate compounds can be carried out by methods known in the art, for example, assays involving using cells such as wild-type cells, cancer cells and/or transgenic liver cells. Representative assays for observing Wnt/FZD signaling, FZD and Wnt expression and cancer cell mobility, and useful cells that can be used in these assays are described in the Examples section below.

Alternatively or additionally, methods for testing candidate compounds in vivo can be carried out by means known in the art. For example, the candidate compound(s) can be administered to a mammal, such as a rodent (eg, mouse) or rabbit. Such animal model systems are recognized in the art as a means of testing effective pharmaceutical agents for measuring the pharmaceutical efficacy of pharmaceutical agents in patients, for example human patients. Animals particularly useful for in vivo testing methods include wild-type animals or non-wild-type animals (e.g. mice) that overproduce FZD and/or Wnt polypeptides, e.g., FZD or Wnt transgenes (e.g., FZD7 or Wnt3). Transgenes), and/or non-wild-type animals that produce less FZD8 and/or Wnt11 polypeptides. Other animals useful for in vivo testing methods include those that have been bred to progress to liver cancer cells. Mice that are particularly useful as any transgenic mice undergoing liver cancer are described in the Examples section below, which are included in the present invention.

In conventional in vivo assays, animals (eg, wild-type or transgenic mice) are administered a dose of a candidate compound via any suitable route of administration (eg, injection). Conventional methods and criteria can then be used to observe whether the animal exhibits the desired activity. If necessary, the results obtained in the presence of the candidate compound can be compared with the results obtained in control animals not treated with the test compound.

Medicinal chemistry

Once the desired compound (or agent) has been identified, derivatives of the compound can be produced to be used in further rounds of testing according to standard principles of medicinal chemistry. Derivatives can be screened for improved pharmacological properties such as efficacy, pharmacokinetics, stability, solubility and extinction rate. The part that is involved in the activity of a compound in the assay described above can be demonstrated by observing the structure-activity relationship (SAR) commonly performed in the art. Those skilled in the pharmaceutical chemistry field have been able to modify the candidate compound or moiety present on the agent to determine how this modification affects the efficacy of the compound or agent, thereby producing a derivative with increased efficacy. See, eg, Nagarajan et al. (1988) J. Antibiot. 41:1430-8]. In addition, if the biochemical target of the compound (or agent) is known or determined, the target and the structure of the compound can inform the design and optimization of the derivative. Molecular-level modeling software for this purpose is commercially available [eg, Molecular Simulations, Inc.].

IV. Antibody

The invention features, for example, purified or isolated antibodies that specifically bind to FZD and/or Wnt polypeptides, i.e. anti-FZD and anti-Wnt antibodies. When an antibody binds to an antigen, but recognizes and binds to other molecules in the sample to a lesser extent (e.g., hardly recognizes and does not bind), the antibody is associated with a specific antigen such as FZD7 and/or It is said to "specifically bind" to the FZD8 polypeptide. Antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ fragments, and molecules produced using Fab expression libraries.

Any type of antibody included in the present invention is, for example, a polyclonal anti-Wnt3 antibody described in the Examples section below. Methods for producing polyclonal antibodies are well known to those skilled in the art.

As used herein, the term "antibody" refers to one or more, for example, two heavy chain (H) variable regions (abbreviated herein as VH), and one or more, for example, two light chain (L) variable regions ( Herein, it means a protein comprising abbreviated as VL). The VH and VL regions may also be subdivided into hypervariable regions, ie "complementarity determining regions (CDRs)", regions that are scattered while retaining more conservative regions, ie "framework regions (FR)". The framework region and the range of CDRs have already been defined [Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917]. Each of the VH and VL consists of 3 CDRs and 4 FRs, which are arranged in the following order in the direction of amino terminal → carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Anti-FZD or anti-Wnt antibodies may also further comprise heavy and light chain constant regions to form heavy and light chain immunoglobulin chains, respectively. The antibody may be a tetramer consisting of two heavy chain immunoglobulin chains and two light chain immunoglobulin chains, wherein the heavy and light chain immunoglobulin chains are interconnected, for example by disulfide bonds. The heavy chain constant region consists of three domains, namely, CH1, CH2, and CH3. The light chain constant region consists of one domain, that is, CL. The variable portions of the heavy and light chains include a binding domain that interacts with an antigen. The constant region of an antibody typically mediates the binding of the antibody to host tissues or factors, such as various immune system cells (eg effector cells) and the first component (C1q) of a conventional complement system.

The terms "FZD binding fragment" and "Wnt binding fragment" of an antibody refer to one or more fragments of a full-length antibody, or portions thereof, each retaining the ability to specifically bind to an FZD or Wnt polypeptide. Examples of the polypeptide binding fragment of the antibody include (i) a Fab fragment, that is, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragment, that is, a divalent fragment comprising two Fab fragments joined by disulfide bonds at the hinge portion; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al, (1989) Nature 341:544-546); And (vi) an isolated complementarity determining region (CDR), but is not limited thereto. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by individual genes, this domain uses a recombination method, so that these domains are combined into a single protein chain (ie, the VL and VH regions are paired to form a Molecules (known as single chain Fv (scFv); e.g. Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883). These single chain antibodies are also included in the terms "FZD binding fragment" and "Wnt binding fragment" of the antibody. The antibody fragments can be obtained using conventional techniques known to those skilled in the art.

In order to prepare an antibody, a polypeptide (or an antigenic fragment of this polypeptide (e.g., a fragment of a polypeptide that is determined to be antigenic according to criteria such as the presence of a large number of charged residues) or an analog of this polypeptide), e.g., Recombinant or peptide synthesis techniques [eg, Solid Phase Peptide Synthesis (homologous); Polypeptides produced by Ausubel et al. (homologous)] can be used. In general, the polypeptide can be coupled to a carrier protein such as KLH (Ausubel et al., (homologous)), mixed with an adjuvant, and also injected into this host mammal. A “carrier” is a substance that stabilizes and/or assists or enhances the immunogenicity or mobility of a bound molecule. For example, FZD or Wnt proteins or fragments thereof can be prepared using standard techniques of PCR and cloned into a pGEX expression vector (Ausubel et al., (homology)). Fusion proteins can be expressed in E. coli as described in Ausubel et al., (homologous), and purified using a glutathione agarose affinity matrix.

Typically, various host animals are injected with FZD and/or Wnt polypeptides. Examples of suitable host animals include rabbits, mice, guinea pigs and rats. Various adjuvants can be used to increase the immune response depending on the type of host, such as frunt (complete and incomplete adjuvant) adjuvant, adjuvant inorganic gel such as aluminum hydroxide, and surface-activating substances such as For example, lysolecithin, polyol of pluronic acid, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin, dinitrophenol, BCG (Bacille Calmette-Guerin) and Corynebacterium parbium parvum ). Through this method, it is possible to produce polyclonal antibodies, that is, heterologous populations of antibody molecules derived from the serum of an immunized animal. The antibody can be purified from blood obtained from a host animal, for example, by affinity chromatography (a method using a resin immobilized with FZD and/or Wnt polypeptide antigen).

The invention also includes anti-FZD monoclonal antibodies and anti-Wnt monoclonal antibodies. Monoclonal antibodies (mAbs), which are homogeneous populations of antibodies specific for a particular antigen, can be prepared using standard hybridoma techniques with FZD or Wnt polypeptides (e.g., Kohler et al., Nature, 256:495. , 1975; Kohler et al., Eur. J. Immunol, 6:511, 1976; Kohler et al., Eur. J. Immunol, 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hvbridomas, Elsevier, NY, 1981; Ausubel et al., (Ibid)].

Typically, monoclonal antibodies are any technique that allows antibody molecules to be produced by continuous cell lines in culture, for example, a technique disclosed in Kohler et al., Nature, 256:495, 1975, and US Pat. No. 4,376,110. ; Human B-cell hybridoma technology [Kosbor et al., Immunology Today, 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026, 1983]; And EBV-hybridoma technology [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1983]. Such antibodies may be of any immunoglobulin group, such as IgG, IgM, IgE, IgA, IgD, and any subgroups thereof. Hybridomas producing the mAb of the present invention can be cultured in vitro or in vivo.

Once produced, polyclonal or monoclonal antibodies are cognitively tested, for example in immunoassays using standard techniques as disclosed in Ausubel et al. (homologous), such as Western blot or immunoprecipitation assays. It can be tested for the ability to specifically recognize FZD or Wnt polypeptides. Antibodies that specifically bind to FZD or Wnt polypeptides (eg, FZD7, FZD8, Wnt3, Wnt8b and/or Wnt11) are useful in the present invention. For example, such antibodies can be used in immunoassays that detect and/or modulate FZD/Wnt signaling in a sample, e.g., a tissue sample, (e.g., treat cancer, e.g. liver cancer). I can.

Alternatively or additionally, monoclonal antibodies can be produced by recombinant methods, eg, Ladner et al., US Pat. No. 5,223,409; Kang et al. International Patent Publication WO 92/18619; Dower et al. International Patent Publication WO 91/17271; Winter et al. International Patent Publication WO 92/20791; Markland et al. International Patent Publication WO 92/15679; Breitling et al. International Patent Publication WO 93/01288; McCafferty et al. International Patent Publication WO 92/01047; Garrard et al. International Patent Publication WO 92/09690; Ladner et al. International Patent Publication WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol. Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; And Barbas et al. (1991) PNAS 88:7978-7982].

Anti-FZD antibodies and anti-Wnt antibodies are entirely human antibodies [e.g., antibodies produced in mice genetically engineered to produce antibodies from human immunoglobulin sequences], or non-human antibodies such as rodents (Mouse or rat), rabbit, horse, cow, goat, primate (eg, monkey), camel, donkey, pig or bird antibodies.

Anti-FZD antibodies and anti-Wnt antibodies may be those in which the variable region or part of the antibody, such as CDRs, is produced in an organism other than human, such as a rat or mouse. Anti-FZD antibodies and anti-Wnt antibodies may also be, for example, chimeric antibodies, CDR-grafted antibodies or humanized antibodies. Anti-FZD antibodies and anti-Wnt antibodies are also produced in non-human organisms, e.g., rats or mice, and then, for example, if a modification occurs in the variable framework or constant region, the immunogenicity in the human body will be reduced. May be.

Technology developed for the production of "chimeric antibodies" [Morrison et al., Proc. Natl. Acad. Sci., 81:6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452, 1984] can be used to splicing genes derived from mouse antibody molecules with suitable antigen specificity and genes derived from human antibody molecules with suitable biological activity. Chimeric antibodies are molecules that have different parts derived from different animal species, such as a variable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques disclosed for the production of single chain antibodies (US Pat. Nos. 4,946,778 and 4,946,778 and 4,704,692) can be applied to produce single chain antibodies specific for FZD or Wnt polypeptides. Single-chain antibodies are formed by linking the light and heavy chain fragments of the Fv region through amino acid bonds.

Antibody fragments that recognize and bind to specific epitopes can be produced by known techniques. For example, such fragments would have reduced the disulfide bonds of, for example, F(ab') ₂ fragments [fragments that can be produced when antibody molecules are digested with pepsin] and Fab fragments [F(ab') _{2 fragments].} It may include a fragment that can be produced when]. Alternatively, the Fab expression library can be constructed to allow rapid and easy identification of monoclonal Fab fragments possessing the desired specificity [Huse et al., Science, 246:1275, 1989].

Polyclonal and monoclonal antibodies (or fragments thereof) that specifically bind to FZD and/or Wnt polypeptides can be used, for example, to detect the expression of FZD and/or Wnt in various tissues of a patient. For example, FZD7 and/or FZD8 polypeptides can be detected in conventional immunoassays of biological tissues or extracts. Examples of suitable assays include, but are not limited to, Western blotting, ELISA, radioimmunoassay, and the like.

V. Pharmaceutical composition

Any pharmaceutically active compound, agent, nucleic acid, polypeptide or antibody (all of which are referred to herein as “active compounds”) can be incorporated into a pharmaceutical composition. Such compositions usually contain an active compound and a pharmaceutically acceptable carrier. As the "pharmaceutically acceptable carrier", solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like suitable for the method of pharmaceutical administration may be included. Supplementary active compounds may also be incorporated into the present composition.

Pharmaceutical compositions can be formulated to suit the intended route of administration. Examples of routes of administration include enteral (e.g., oral or rectal) administration and parenteral administration, such as intravenous (e.g., portal vein), intradermal, subcutaneous, transdermal, transmucosal and intrapulmonary administration. . Administration can be made directly to the liver, for example by injection or topical administration during surgery. Solutions or suspensions used for injection may contain the following components: sterile diluents such as water for injection, saline solution, nonvolatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; Antimicrobial agents such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate and osmotic pressure modifiers such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules made of glass or plastic, disposable syringes or multiple dose vials.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersants and sterile powders used to prepare sterile injectable solutions or dispersions on the fly. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that it is readily injectable. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria or fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, if a coating that maintains an appropriate particle size during dispersion, for example, lecithin and a surfactant, is used, the fluidity can be properly maintained. Various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. can be used to prevent the activity of microorganisms. In many cases, it will be desirable to include isotonic agents such as sugars, polyalcohols such as mannitol or sorbitol and sodium chloride. If an agent that sustains absorption for a long time, for example, aluminum monostearate and gelatin, is added to the present composition, the absorption time of such an injectable composition can be prolonged.

Sterile injection solutions can be prepared by adding an active compound in a necessary amount in an appropriate solvent, adding any one of the ingredients listed above or a combination of these ingredients, followed by sterilization filtration. In general, dispersions are prepared by adding the active compound to a sterile vehicle, i.e., a basic dispersion medium and a vehicle containing the necessary components of the above-listed components. In the case of a sterilized powder for preparing a sterile injectable solution, preferred manufacturing methods include a vacuum drying method and a freeze drying method, and through these methods, the active ingredient powder and the desired optional additional ingredients can be obtained from the previously sterilized filtered solution. have.

Compositions for oral administration generally contain an inert diluent or an edible carrier. For oral administration of therapeutic agents, the active compounds can be incorporated with excipients and can also be used in the form of tablets, troches or capsules, for example gelatin capsules. Compositions for oral administration may also be prepared using a carrier solution for oral washing. Pharmaceutically acceptable binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches, and the like may contain any of the following ingredients, or compounds having similar properties: Binders such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch; Lubricants such as magnesium stearate or sterotes; Glidants such as colloidal silicon dioxide; Sweetening agents such as sucrose or saccharin; Or flavoring agents such as peppermint, methyl salicylate or orange flavor.

For administration by inhalation, the compounds of the present invention are delivered in the form of aerosol sprays or nebulizers sprayed from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide.

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for making the serous membrane permeable are used in the formulation. These penetrants are generally known in the art, and examples thereof include penetrants for transmucosal administration, detergents, bile salts and fucidic acid derivatives. Transmucosal administration can be carried out by nasal sprays or suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides). For transdermal administration, the active compound is formulated in the form of an ointment, salve, gel or cream, as is generally known in the art.

In one embodiment, the active compound is prepared with a carrier, such as a controlled release formulation, such as an implant and a micro-enclosed delivery system, that prevents the rapid disappearance of the compound in the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyhydric anhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. How to prepare such formulations will be understood by one of skill in the art. This material is also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. This suspension can be prepared according to methods known to the person skilled in the art, for example as disclosed in U.S. Patent No. 4,522,811.

It may be advantageous to formulate a composition for oral or parenteral administration in a dosage unit form that allows easy administration and can be administered uniformly. Dosage unit form, as used herein, is meant to mean a physically separate unit suitable for single administration to an individual to be treated; Each unit contains, together with the required pharmaceutical carrier, an amount of active ingredient calculated to produce the desired therapeutic effect.

The toxicity and therapeutic efficacy of these compounds are, for example, in standard pharmaceutical methods of measuring LD50 (a dose that is lethal to 50% of the population) and ED50 (a dose that is therapeutically efficacious in 50% of the population). Therefore, it can be determined in a cell culture medium or in an experimental animal. The dose ratio between the toxic effect and the therapeutic effect is called the therapeutic index, and this therapeutic index can be expressed as a ratio of LD50/ED50. Compounds with a high therapeutic index are preferred. Compounds that exhibit toxic side effects may be used, but in such cases care should be taken in designing delivery systems that target compounds at affected tissue sites, such as the liver, so as to reduce side effects by minimizing possible damage to healthy cells.

Data obtained through cell culture assays and animal studies can be used to formulate dosage ranges for humans. It is preferred that the dosage of such compounds be within the range of the circulating concentration including the ED50, with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the methods of the invention, the therapeutically effective dosage can be initially estimated through cell culture assays. Dosage is within the animal model to reach a range of concentrations in the circulating plasma, i.e., the IC50 measured in the cell culture (i.e., the concentration of the test compound corresponding to half of the concentration capable of maximally suppressing symptoms). Can be formulated. This information can be used to more accurately determine the effective dose in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

As used herein, the terms "effective amount" and "effective for treatment" are used for a period of time (e.g., short-term or long-term administration and periodic or continuous administration), which may produce an intended effect or physiological result. It refers to the amount or concentration of a compound that is effective within the dosage range. For the compounds disclosed herein, for example, the effective amount (i.e., effective dosage) of the polypeptide ranges from about 0.001 to 500 mg, such as about 0.01 to 50 mg, such as about 0.1 to 20 mg per kg of body weight. to be. The polypeptide may be administered once a week for about 1-10 weeks, for example 2-8 weeks, about 3-7 weeks, or over a period of about 4, 5, or 6 weeks. Those of skill in the art will be aware of any factors, e.g., the type of patient being treated, the severity of the disease or condition, prior treatment history, the patient's overall health and/or age, and the administration required to effectively treat the patient. You will understand that it affects quantity and time. In addition, as a method of treating a patient with a therapeutically effective amount of a compound, a single therapy may be included, or preferably a continuous therapy may be included.

For antibodies, partial human antibodies and fully human antibodies have a longer half-life in the human body than other antibodies. Therefore, the dosage and frequency of administration can be further reduced. For example, lipidation can be used to stabilize antibodies and enhance absorption and tissue penetration. A method for lipidating antibodies is disclosed in Cruikshank et al. (1997), J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193.

If the compound is a small molecule, a typical dosage includes the small molecule in the amount of mg or µg per 1 kg of individual or sample weight [for example, about 1 µg/kg to about 500 µg/kg, about 100 Μg/kg to about 5 mg/kg, or about 1 μg/kg to about 50 μg/kg]. In addition, it will be appreciated that the appropriate dosage of a small molecule will depend on the amount of expression to be adjusted or the efficacy of the small molecule on the activity. When one or more of these small molecules are administered to an animal (e.g., a human) to modulate the expression level or activity of an FZD or Wnt polypeptide or nucleic acid, a specialist, veterinarian or researcher may, for example, initially administer relatively small doses. It is prescribed in doses, and later the dosage can be increased until a suitable response is finally achieved. In addition, the dosage level specific to any particular animal subject may depend on a number of factors, such as the activity of the particular compound used, age, weight, general health, sex and dietary habits of the subject, time of administration, route of administration, It will depend on the rate of excretion, the administration of any combination of drugs, and the level of expression or activity to be adjusted.

Nucleic acid molecules (eg, FZD7, FZD8, Wnt3, Wnt8b and/or Wnt11 DNA) can be inserted into a vector and used as a vector for gene therapy. Vectors for gene therapy can be administered to an individual, for example, intravenous injection, topical administration (eg, US Pat. No. 5,328,470) or stereotactic injection (eg, Chen et al. (1994) Proc. Natl. Acad. Sci.USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector may include the gene therapy vector in an acceptable diluent, or may include a sustained release matrix embedded with a gene delivery vehicle. Alternatively, where a complete gene transfer vector, eg, a retroviral vector, can be produced intact from a recombinant cell, the pharmaceutical preparation may comprise one or more cells producing the gene delivery system. Representative structures that can be effectively used in gene therapy are described in the Examples section below.

The pharmaceutical composition may be included in a container, pack or dispenser with instructions for administration.

VI. Cancer and its treatment methods

The term "cancer" refers to an animal cell that retains the ability to grow spontaneously. Examples of such cells include cells exhibiting an abnormal state or pathology characterized by rapid growth and proliferation of cells. The term is meant to include the growth state of cancer cells, regardless of histological type or invasion stage, and includes, for example, tumors, carcinogenic processes, metastatic tissues and malignantly deformed cells, tissues or organs. In addition, malignant tumors of various organ systems, such as respiratory, cardiovascular, kidney, genital, hematopoietic, nervous, liver, gastrointestinal and endocrine systems; And malignant tumors such as most colon cancer, kidney-cell carcinoma, prostate cancer and/or testicular cancer, non-small cell cancer of the lung, adenocarcinoma including small intestine cancer and esophageal cancer. "Naturally occurring" cancer includes any cancer that is not experimentally induced by injecting cancer cells into an individual, for example, cancer that occurs spontaneously, cancer that occurs when a patient is exposed to a carcinogen, and transgenics. There are cancers caused by insertion of oncogenes or loss of function of tumor suppressor genes, and cancers caused by infections such as viral infections. The term "carcinoma" is known in the art as being a malignant tumor of an epithelial or endocrine tissue. The term includes carcinosarcoma, which includes malignant tumors consisting of cancerous tissues and sarcoma tissues. "Adenocarcinoma" means a carcinoma derived from glandular tissue or a tumor cell that forms a recognizable glandular structure. The term "hepatocellular carcinoma (HCC)" refers to cancer that occurs in liver cells, ie, the major cell types that make up the liver.

Throughout this specification, the term "patient" refers to animals, that is, humans or non-humans, rodents or non-rodents, and refers to animals to be treated according to the method of the present invention. Veterinary and human clinical trials are also considered. The term "patient" includes, for example, birds, reptiles, amphibians and mammals such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cattle, horses, cats, It also includes dogs, sheep and goats. Preferred individuals are humans, farm animals, and pets such as cats and dogs. As used herein, the term "treatment (method)" means prolonging the life of a patient suffering from a disease condition, for example, cancer, or suppressing, alleviating the power of cancer, and delaying the occurrence of cancer.

Cancers that can be treated using the methods and compositions of the present invention include, for example, liver cancer, gastric cancer, colon cancer, rectal cancer, oral cancer/pharyngeal cancer, esophageal cancer, laryngeal cancer, pancreatic cancer, lung cancer, small intestine cancer and bile duct cancer.

Since treatment can be initiated prophylactically, primarily before the onset of a tumor-related prognosis, individuals believed to be at risk of developing cancer may particularly benefit from the present invention. An individual "at risk", for example, an individual exposed to a carcinogen, e.g., a sensitive individual, consumes the carcinogen at a level that is statistically determined to trigger the onset of cancer, e.g., inhalation and/or Includes ingested individuals. Individuals who have been exposed to a virus such as a hepatitis virus such as hepatitis B virus (HBV) are also included. Individuals who have been exposed to ultraviolet light, or who have been at risk factors due to environmental, occupation, and/or genetics, and who exhibit signs of precancerous conditions are also included. Similarly, individuals in the early stages of cancer or in the process of metastasis (i.e., the presence of one or a few abnormal cells in the individual's body or in a specific area of the individual's tissue) will benefit from this prophylactic treatment. I can.

The person skilled in the art will, using the methods disclosed herein, optionally additional methods, e.g., assessing the patient's medical history, performing other diagnostic tests, and/or using imaging techniques, a specialist (or suitable for diagnosing a patient). It will be understood from a veterinarian) that a patient may have developed or are at risk of developing cancer.

One technique for treating patients with or at risk of developing cancer is to modulate Wnt/FZD signaling in the patient. The goal of this is to accelerate signaling if the signaling is too slow, and slow signaling if the signaling is too fast. Modulation of Wnt/FZD signaling can be divided into two basic classes: reducing (i.e., inhibition, e.g., extinction) Wnt/FZD signaling when activity is insufficient or inactive. And, increasing (ie, supplementing or providing) Wnt/FZD signaling. Whether Wnt/FZD signaling should be inhibited or increased depends on the intended purpose. Wnt/FZD signaling can be modulated using active compounds (eg, anti-Wnt antibodies, siRNAs, candidate compounds and/or anticancer agents), as described herein. For example, by reducing the expression level of FZD7 and/or Wnt3 and/or interfering with the interaction between FZD7 and its ligands (e.g., Wnt3, 8b and/or 11), reducing Wnt/FZD signaling activity The compound that makes it can be used, for example, as a therapeutic agent for cancer, such as liver cancer. For example, compounds that increase the activity by increasing the expression level of FZD8 can also be used, for example, as a therapeutic agent for cancer, such as liver cancer.

Reduction of Wnt/FZD signaling

Methods known in the art to reduce the expression of certain proteins in the body of a patient can be used to reduce Wnt/FZD signaling. For example, antisense nucleic acids effective to inhibit the expression of endogenous FZD or Wnt genes, such as the FZD7 gene or Wnt3 gene, can be used. As used herein, the term "antisense oligonucleotide" or "antisense" refers to DNA under physiological conditions, i.e. DNA comprising a specific gene, or to the mRNA transcript of the gene, and the transcription of this gene and/or It refers to an oligonucleotide that inhibits the translation of, that is, an oligoribonucleotide, an oligodeoxyribonucleotide, a modified oligoribonucleotide or a modified oligodeoxyribonucleotide.

Antisense molecules are designed to interfere with the transcription or translation of a target gene (eg, a gene encoding FZD7 or Wnt3, 8b or 11) as it hybridizes with the target gene or transcript. The antisense nucleic acid may include a nucleotide sequence complementary to the entire FZD or Wnt RNA or only a portion of the RNA. On the other hand, the antisense nucleic acid needs to be long enough to effectively hybridize with FZD or Wnt RNA. Therefore, the minimum length is about 12-25 nucleotides. On the other hand, if the length is increased to more than about 150 nucleotides, the efficacy of inhibiting translation can be increased by that limit, while introducing antisense nucleic acids into target cells can be much more difficult. Therefore, a suitable length as an antisense nucleic acid may be about 15 to about 150 nucleotides, for example 20, 25, 30, 35, 40, 45, 50, 60, 70 or 80 nucleotides. Antisense nucleic acids may be complementary to the coding region of FZD or Wnt mRNA, or the 5'or 3'non-coding region of FZD or Wnt mRNA, or both. One method is to design antisense nucleic acids to be complementary to sites present at both the translation initiation sites of FZD or Wnt mRNA.

Based on the sequences disclosed herein, one of ordinary skill in the art can easily select and synthesize any of a number of suitable antisense molecules used in accordance with the present invention. For example, by providing a "gene walk" comprising a plurality of oligonucleotides complementary to FZD or Wnt mRNA and having a length of 15 to 30 nucleotides extending in the length direction thereof, to inhibit FZD or Wnt expression Can be tested. Optionally, a gap of 5 to 10 nucleotides exists between the oligonucleotides, allowing the number of oligonucleotides to be synthesized and tested to be reduced.

Antisense nucleic acids can be chemically synthesized, for example, using a commercially available nucleic acid synthesizer according to the instructions of the marketplace. Alternatively, antisense nucleic acids can be prepared using recombinant DNA technology. Antisense nucleic acids can only be incorporated into naturally occurring nucleotides. Alternatively, the antisense nucleic acid can be incorporated into variously modified nucleotides or nucleotide analogues, increasing its half-life in vivo, or increasing the stability of the duplex formed between the antisense molecule and its target RNA. . Examples of nucleotide analogues include phosphorothioate derivatives and acridine-substituted nucleotides. When designating targets and sequences, methods for designing and producing suitable antisense molecules are known to the person skilled in the art. For antisense nucleic acids, see Goodchild, "Inhibition of Gene Expression by Oligonucleotides," in Topics in Molecular and Structural Biology, Vol. 12: Oligodeoxynucleotides (Cohen, ed.), MacMillan Press, London, pp. 53-77 (1989).

Delivery of antisense oligonucleotides can be carried out by any method known to those skilled in the art. For example, when delivering antisense oligonucleotides for cell culture and/or extracellular work, they are delivered by standard methods such as liposome methods or simply by adding membrane permeable oligonucleotides.

Delivery of an antisense oligonucleotide to an in vivo method can be, for example, by a method of local injection of an antisense oligonucleotide at a selected location, such as the liver. This method has already been demonstrated for inhibition of the growth of psoriasis and inhibition of cytomegalovirus. See, for example, Wraight et al., (2001). Pharmacol Ther. 90(1):89-104; Anderson et al., (1996) Antimicrob Agents Chemother 40: 2004-2011; And Crooke et al., (1996) J Pharmacol Exp Ther 277: 923-937.

Similarly, RNA interference (RNAi) technology can be used to inhibit FZD or Wnt expression in addition to or as an alternative to antisense technology. For example, small interfering RNA (siRNA) duplexes directed against FZD or Wnt nucleic acids can be synthesized and used to block the expression of the encoded protein(s). Representative Wnt3 siRNAs are described in the Examples section below.

Another approach to inhibiting Wnt/FZD signaling is a compound that binds to an FZD polypeptide (e.g., FZD7 polypeptide) to a patient, e.g., a candidate compound or anticancer agent and/or its binding partner (e.g., Wnt3, 8b And/or 11) and preventing an interaction between the two substances. Such compounds and agents bind, for example, to FZD polypeptides (e.g., CRD domains of FZD polypeptides) in a form that prevents protein-to-protein interactions, and/or Wnt polypeptides (e.g., binding domains of Wnt polypeptides). Can be combined with These candidate compounds and anticancer agents can be identified through the screening method disclosed herein. Examples of compounds capable of binding to Wnt polypeptides, such as Wnt3, 8b and/or 11, include the FZD7 receptor or truncated form thereof, as described in the Examples section below, and anti-Wnt antibodies (or FZD-binding fragments thereof) examples. For example, there is an anti-Wnt3 antibody.

Another approach to inhibiting Wnt/FZD signaling is to give the patient a vector (e.g., a gene therapy vector) encoding a mutant (e.g., truncated) form of the FZD receptor, e.g., the FZD7 receptor. And administering. Expression of the receptor mutant by patient cells (ie, cells containing constructs) can interfere with intracellular Wnt/FZD signaling. For example, constructs encoding secreted and soluble forms of the FZD receptor (eg, FZD7 receptor) can be used. When these structures are expressed by a target cell, the target cells secrete the FZD receptor that will bind to the Wnt polypeptide in a soluble form. As a result, the Wnt polypeptide can bind to the original FZD receptor on the cell surface. There will be no. Alternatively or additionally, constructs encoding the membrane-bound, but inactive form, the FZD receptor (i.e., a mutant FZD receptor that cannot perform some of the actions carried out by the corresponding wild-type FZD receptor) can be used. . When these constructs are expressed by target cells, they can bind to the Wnt polypeptide or interfere with Wnt/FZD signaling through an internal mechanism that does not involve the Wnt polypeptide. Another method of inhibiting Wnt/FZD signaling involves administering to the patient a vector encoding a Wnt11 polypeptide (e.g., a vector for gene therapy), wherein the Wnt11 polypeptide is of the Wnt pathway in a typical HCC. It is an inhibitory factor. The vector may be derived from a non-replicating linear or circular DNA or RNA vector, or may be derived from a spontaneously replicating plasmid or viral vector. Methods for constructing an appropriate expression vector are known in the art, and useful materials for this are commercially available.

Increased Wnt/FZD signaling

Novel or complementary Wnt/FZD signaling is provided by increasing the expression of FZD polypeptides (e.g., FZD8 polypeptides) and/or Wnt polypeptides (e.g., Wnt3 polypeptides) in vivo, i.e. in the body of a patient. Can be. For example, the FZD or Wnt polypeptide is an organism by expressing a nucleic acid construct containing a nucleotide sequence encoding an FZD polypeptide (e.g., FZD8 polypeptide) and/or a Wnt polypeptide (e.g., Wnt3 polypeptide) in an organism cell. For example, it can be produced directly in the human body. Any expression vector suitable for transfecting the cells of the organism of interest can be used for this purpose.

VII. Transgenic animals

The present invention also features a transgenic animal that develops liver cancer and overexpresses FZD7 in liver cells. These animals represent a model system for liver cancer research and the development of therapeutics capable of modulating Wnt/FZD signaling and treating cancer.

Transgenic animals include, for example, farm animals (pigs, goats, sheep, cattle, horses, rabbits, etc.), rodents (e.g. rats, guinea pigs and mice), non-human primates (e.g., baboons). Monkeys, monkeys and chimpanzees), and pets (eg, dogs and cats).

Transgenes can be introduced into animals using any technique known in the art to create a founder line of transgenic animals. Examples of such techniques include pronuclear microinjection (US Pat. No. 4,873,191); A method of transporting genes to germ cells via retrovirus (Van der Putten et al., Proc. Natl. Acad. Sci, USA 82:6148, 1985); Gene targeting to embryonic stem cells (Thompson et al., Cell 56:313, 1989); And electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803, 1983), but is not limited thereto. The method disclosed in Yang et al. (Proc. Natl Acac. Sci. USA 94:3004-3009, 1997) is particularly useful.

For a more detailed understanding of techniques that can be used to produce and evaluate transgenic animals, the person skilled in the art is described in Intl. Rev. Cytol. 115:171-229, 1989, see also Hogan et al. Manipulating the Mouse Embryo, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1986; Krimpenfort et al. (Bio/Technology 9:86, 1991), Palmiter et al. (Cell 41:343, 1985), Kraemer et al. (Genetic Manipulation of the Early Mammalian Embryo, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1985) ), Hammer et al (Nature 315:680, 1985), Purcel and others (Science, 244:1281, 1986), Wagner and others (US Patent No. 5,175,385), and Krimpenfort and others (US Patent No. 5,175,384)] Additional guidance can also be obtained through this program.

[Example]

The invention is demonstrated in part through the following examples, and the following examples are not intended to limit the invention.

Example 1. Identification of natural Wnt ligand for inhibition of HCC growth

Way

Preparation of cell surface HSPG and fractionation by heparin-agarose chromatography

Huh7 cells were cultured in 10% FBS (Sigma, St. Louis, MO) MEM (Mediatech, Herndon, VA, USA) until 70% confluence. 24 hours after incubation, this medium was changed to 0% FBS MEM, and the cells were treated with heparin (50 ug/ml) or not in 0.1% FBS MEM for 12 hours. Cells were washed 5 times with ice-cold PBS, and the cell layer was incubated for 10 minutes on ice with crystallized trypsin (20 ug/ml) in Tris-buffered saline/EDTA. Soybean trypsin inhibitor was added thereto to a final concentration of 100 ug/ml to stop trypsin activity. The reaction was centrifuged at 4° C. for 5 minutes (400 x g) to remove contaminating cells from trypsin-treated cells.

The trypsin-treated cells were subjected to heparin-agarose chromatography. Briefly, trypsin-treated cells were incubated overnight (4° C.) with heparin (4%) agarose beads, and then these beads were collected by centrifugation at 2000 xg for 4 minutes, and then these beads were collected in PBS. Washed with 20 volumes of 0.1 M NaCl. The eluted fractions were collected using 0.25, 0.5, 0.75 and 1.0 M NaCl in PBS.

Two-dimensional gel electrophoresis in gel digestion and peptide mapping

Samples were fractionated from heparin-agarose chromatography by precipitation and rehydration with IPG buffer for isoelectric focusing (IEF). IEF was performed using a ZOOM IPGRunner (Invitrogen™, Carlsbad, Calif.) according to the manufacturer's protocol. After rehydrating the zoom strip (pH3-10) with the sample overnight, a step voltage ramping method was performed as follows: 200 V, 20 minutes → 450 V, 15 minutes → 750 V, 15 Min → 2000 V, 120 min. The focused gel was subjected to SDS-PAGE (two-dimensional electrophoresis) using a zoom gel (Invitrogen). After electrophoresis, the gel was stained using a SilverQuest Silver Staining kit (Invitrogen).

The protein spot was excised from the silver-stained gel, bleached and dried, and then enzymatically digested using a sequence-grade modified trypsin (Promega, Madison, Wis.). Desalting the trypsin cutting peptide and it was concentrated to jiptip _C18 (ZipTip _C18) (methoxy situ Bedford, Massachusetts State, Millipore). The concentrated trypsin-treated peptide was added to the SEND ProteinChip, and peptide mapping was performed with PBSII (Ciphergen, Fremont, Calif.). Peptide mass fingerprinting was performed using a database search tool (http://prospector.ucsf.edu) called MS-Pete of a program called Protein Prospector. Based on the results of measuring the location of the spot in the 2-D gel, an initial search was performed for a number of restriction sites: species = homo sapiens, pI range = 6 to 9.5, mass range = 35 to 50 kDa.

RT-PCR analysis

Whole cell RNA was extracted from HepG2, Hep3B, Huh7 and focus cell lines using TRIzol® reagent (Invitrogen). The first chain cDNA was converted into 250 ng of total RNA with random hexamer and AMV RT using a first chain cDNA synthesis kit for RT-PCR (AMV) (Roche Diagnostics, Indianapolis, IN) in 20 μl of the reaction mixture. Synthesized. Using 50 ng of cDNA and High Fidelity PCR Master (Roche Diagnostics), PCR was performed in a thermal cycle reactor (MJ Research Inc., Waltham, Mass.). Primer pairs for each Wnt ligand are listed in Table 1 below. The final concentration of each primer was 250 nM. After initially denaturing at 94°C for 4 minutes, the reaction was repeated 35 times according to the following temperature program: 94°C, 30 seconds → 55°C, 30 seconds → 72°C, 1 minute → final amplification step; 72° C., 10 minutes. The amplified product was analyzed on a 2% agarose gel stained with ethidium bromide.

qRT-PCR assay

As described above, total RNA and RT reactants were extracted from liver tissue and HCC cell lines. SYBR Green PCR Master Mix (Foster City, CA, Applied Biosystems), a mixture consisting of 400 nM of each primer and 5 ng of cDNA (equal total RNA), i-Cycler iQ multicolor real-time PCR detection system By performing qRT-PCR using Multi-Color Real Time PCR Detection System; Hercules, Calif., Bio-Rad), mRNA expression levels of Wnt3, Wnt11 and FZD7 were measured from unknown samples. Thermal cycling conditions were initially 10 minutes at 95°C → 15 seconds at 95°C (40 cycles) → 30 seconds at 60°C → 30 seconds at 72°C. Primer sequences for Wnt3, Wnt11, FZD7 and 18S rRNA were as follows:

(1) Wnt3, 5'-ACTTCGGCGTGTTAGTGTCC-3' (forward) (SEQ ID NO: 68) and 5'-CATTTGAGGTGCATGTGGTC-3' (reverse) (SEQ ID NO: 69);

(2) Wnt11, 5'-TTCCGATGCTCCTATGAAGG-3' (forward) (SEQ ID NO: 70) and 5'-AGACACCCCATGGCACTTAC-3' (reverse) (SEQ ID NO: 71);

(3) FZD7, 5'-GCCGCTTCTACCACAGACT-3' (forward) (SEQ ID NO: 72) and 5'-TTCATACCGCAGTCTCCCC-3' (reverse) (SEQ ID NO: 73);

(4) 18SrRNA, 5'-GGACACGGACAGGATTGACA-3' (forward) (SEQ ID NO: 74) and 5'-ACCCACGGAATCGAGAAAGA-3' (reverse) (SEQ ID NO: 75).

The size of the amplicon was 130 bp for Wnt3, 133 bp for Wnt11, 54 bp for FZD7, and 50 bp for 18SrRNA. After visualizing the PCR product by 1.5% agarose gel electrophoresis and ethidium bromide, the PCR product was excised and cloned into a pCR2.1 vector (Invitrogen). Sequencing was performed in both directions using the T7 forward primer and the M13 reverse primer. After confirming the nucleotide sequence, each PCR product cloned into the pCR 2.1 vector was diluted 10-fold to prepare a real-time PCR standard. After measuring the Ct value, the number of copies of Wnt3, Wnt11 and FZD7 mRNA among unknown samples was quantified by normalizing to 18SrRNA by comparing with a standard curve for each gene. The experiment was conducted twice.

Preparation of plasmid and anti-Wnt3 Ab

Since 22 amino acids present in the original plasmid have been deleted at the C-terminus of the human Wnt3 plasmid, the sequence of human Wnt3 was cloned into pcDNA™3.1/myc-His A (Invitrogen) through the sites of HindIII and EcoRII restriction enzymes ] Based on the PCR amplification process was performed three times to amplify the full-length sequence. The primer sequence was as follows:

5'-TAGTTAAGCTTACCATGGAGCCCCACCTGCTC-3' (forward) (SEQ ID NO: 76),

5'-GCAGCTGACGTAGCAGCACCAGTGGAAGATGCAGTG-3' (reverse-1) (SEQ ID NO: 77),

5'-GTAGATGCGAATACACTCCTGGCAGCTGACGTAGCA-3' (reverse-2) (SEQ ID NO: 78),

5'-TGCTTGAATTCCTTGCAGGTGTGCACGTCGTAGATGCGAATACA-3' (reverse-3) (SEQ ID NO: 79).

Rabbit polyclonal antibodies were prepared against a synthetic peptide corresponding to amino acids 259-274 of human Wnt3 ( ²⁵⁹ LRAKYSLFKPPTERDL ^{274) (SEQ ID NO: 80).} The peptide sequence had little homology to other members of the Wnt group or other known proteins. The specificity of the antibody was confirmed by Western blot analysis using an HCC cell line transfected with the myc-tagged Wnt3 plasmid.

Cell culture fluid

Hep3B, Huh7 and focus cell lines were cultured in MEM supplemented with 10% FBS and 1× minimal essential medium nonessential amino acid solution (Sigma). Since a mutation in which the β-catenin gene (31) was deleted occurred in HepG2 cells, these HepG2 cells were excluded. In accordance with the manufacturer's instructions, transfection experiments were conducted at 70% confluence using LipofectAMINE 2000 (Invitrogen) or TransIT-LT1 (Mirus, Madison, Wis.). After transfecting cells with pSUPER8xTOPFLASH or pSUPER8xFOPFLASH (32), Tcf transcriptional activity was analyzed using a luciferase assay system (Promega, Madison, Wis.). Untreated data on luciferase activity were normalized using β-galactosidase activity as a transfection control. The experiment was repeated 3 times to verify the results.

Effect of Wnt3 overexpression in HCC cells

HCC cells were transfected with pcDNA3/myc-His A (control plasmid) or Wnt3 plasmid, pSUPER8xTOPFLASH or pSUPER8xFOPFLASH, and β-galactosidase plasmid. 24 hours after transfection, the cells were incubated in 0% FBS MEM for 24 hours, and then 1% FBS MEM was added to promote culture. Cells were collected 2 hours and 24 hours after the culture was promoted, and the luciferase assay and Western blot assay for Wnt3 and β-catenin were performed.

Effect of anti-Wnt3 antibody

To block the experiment, cells were inoculated into 12-well plates and transfected with pSUPER8xTOPFLASH or pSUPER8xFOPFLASH together with the above-described β-galactosidase plasmid. After subtracting the serum for 24 hours, the cells were incubated with 1% FBS MEM containing anti-Wnt3 Ab or control Ab (10 μg/ml), and cells were collected 24 hours after the incubation as described above. Normal rabbit IgG (Upstate, Waltham, Mass.) was used as a control antibody.

siRNA effect

Control siRNA and Wnt3 siRNA [WNT3-1: 5'-GGAAAAAUGCCACUGCAUC-3' (SEQ ID NO: 81), WNT3-2: 5'-GGAGUGUAUUCGCAUCUAC-3' (SEQ ID NO: 82), WNT3-3: 5'-GGCUUAUCUUUGCACAUGU-3 '(SEQ ID NO:83)] were purchased from Ambion (Austin, Texas), and these were purchased with pSUPER8xTOPFLASH or pSUPER8xFOPFLASH, and β-galactosidase plasmid at a concentration of 10 or 100 nM [TransIT-LT1 or DharmaFECT 4 (using Dharmacon, Chicago, IL)] HCC cells were transfected. 48 hours after transfection, mRNA expression levels of Wnt3 and Tcf transcriptional activities were measured by qRT-PCR and luciferase assays, respectively.

Effect of blocking anti-Wnt3 Ab during wound healing

Focus cells were plated on 6-well plates. Confluent monolayer cells were wounded with the tip of a 200 μl sterile plastic micropipette. Thereafter, the cells were treated with a medium containing anti-Wnt3 Ab or rabbit IgG, and pictures of these cells were taken at different time points.

Western blot analysis

To extract total protein, cells were subjected to lysis buffer (30 mM Tris, pH 7.5, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing proteinase inhibitor (Roche Diagnostics). , 10% glycerol and 2mM EDTA), followed by sonication. Protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL) and BSA (standard). The same amount of protein (150 μg) was separated using 12-15% SDS-PAGE, and this was transferred to a PVDF membrane (PerkinElmer, Wellesley, Massachusetts). The membrane was blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20, which was then subjected to mouse monoclonal antimyc Ab (1:1,000 dilution) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), rabbit Polyclonal anti-Wnt3 Ab (1:200 dilution), mouse monoclonal anti-β-catenin Ab (1:1,000 dilution) (Transduction Laboratories, San Diego, CA), or rabbit polyclonal anti-hsp90 Ab (1:2,000) Dilution) (Santa Cruz Biotechnology Inc.) and incubated overnight at 4°C. After washing with Tris-buffered saline containing 0.1% Tween 20, the membrane was incubated with a secondary HRP antibody (1:10,000 dilution) for 1 hour 30 minutes at room temperature, followed by chemiluminescence image formation Western blot electrophotometry ( PerkinElmer).

Human HCC tissue

Peri-tumor liver tissues matched with 17 pairs of HCCs, but not associated with them, were obtained from South Korea and South Africa. Twelve pairs of samples were obtained from patients of Korean nationality who underwent resection surgery. Of the 12 patients, 9 were male, with a median age of 52 years (range: 22-67 years). 11 patients were positive for the hepatitis B virus surface antigen (HBsAg), and the other 1 patient had no etiology. Seven patients (58%) were in progress of cirrhosis. Five liver tissues were obtained from patients of Korean nationality who had metastasized to the liver and underwent liver resection. These tissues were used as controls for normal liver tissues. Four of the patients were male and the median age was 46 years (range: 37-57 years). As a result of anatomical observation, no pathology was observed in the surrounding liver tissue around the tumor.

Immunohistochemistry for β-catenin

The specimens fixed in formalin and buried in paraffin were immersed in xylene to remove paraffin, and then rehydrated while decreasing the concentration of ethanol. The specimen was immersed in 10 mM sodium citrate buffer (pH 6.0) heated in a microwave oven for 10 minutes, and then cooled to room temperature to reactivate the epitope. The slides were then processed with Universal DakoCytomation LSAB® + Kit, peroxidase (DAKO Corp., Carpinteria, Calif.) according to the manufacturer's instructions. This was blocked by using 3% hydrogen peroxide and endogenous avidin/biotin blocking kit (Endogenous Avidin/Biotin Blocking Kit; Zymed Laboratories Inc., South San Francisco, CA) to block endogenous peroxidase, avidin, and biotin activity. Specimens were incubated overnight at 4° C. with a 1:500 diluted solution of anti-human β-catenin monoclonal Ab. As a negative control, the primary antibody was replaced with PBS. β-catenin expression patterns were classified into two groups depending on whether or not nuclear staining was performed. The cytoplasm of the tumor tissue was stained and compared with the staining result of the tissue around the tumor.

Exon-3 mutation analysis of β-catenin gene

The exon 3 mutation of the β-catenin gene was analyzed using a slightly modified method of Wong et al. (11). Briefly, a 218 bp fragment of exon 3 of the β-catenin gene was amplified from HCC and cDNA (50 ng) of the surrounding tumor liver tissue using the Expand High Fidelity PCR System (Roche Diagnostics). Made it. The primer sequences were 5'-GATTTGATGGAGTTGGACATGG-3' (forward) (SEQ ID NO: 84) and 5'-TGTTCTTGAGTGAAGGACTGAG-3' (reverse) (SEQ ID NO: 85). Thermal cycling conditions were initially 3 minutes at 94°C (denaturation step) → 30 seconds at 95°C (35 cycles) → 30 seconds at 58°C → 30 seconds at 72°C. After visualizing the PCR product, the PCR product was cloned into a pCR 2.1 vector (Invitrogen), and sequenced using a T7 forward primer and an M13 reverse primer. Five or more clones were obtained from each PCR product and sequenced and analyzed.

result

Identification of Wnt ligands in HCC cell lines

Since the Wnt protein is mainly bound to the ECM present on the cell surface, an attempt was made to purify the cell surface HSPG, eg, Wnt protein(s), as a Huh7 cell bound type. Huh7-HSPG produced by trypsin treatment was added to a heparin-agarose affinity resin to perform pre-fractionation. The eluted fraction was subjected to SDS-PAGE, and then compared with the protein bands of the heparin-treated and untreated samples. Observation of the 0.25 M NaCl elution fraction showed a protein band of approximately 45 kDa in the untreated fraction of heparin (data not shown). Since the predicted molecular weight of the Wnt protein corresponds to 35-45 kDa, this protein band was considered to potentially contain the Wnt protein. Two-dimensional electrophoresis was performed to identify this protein band. As shown in FIG. 1A, several silver-stained protein spots having different protein expression levels between the heparin-treated sample and the heparin-free sample were formed. Protein spots with a molecular weight of 35 to 55 kDa and pI 5 to 9.5 were considered to be candidates for the Wnt ligand protein. Since the Wnt protein can be released by treatment with heparin, two upregulated protein spots in untreated samples with heparin were investigated. Protein spots excised from the silver-stained two-dimensional gel were digested in the gel and then peptide mapped by mass spectrometry. As a result of data analysis, it was found that 9 peptides matched the human Wnt11 protein (FIG. 1B). In order to clearly support these observations, the expression of Wnt mRNA in HCC cell lines was observed through RT-PCR using 19 pairs of primers specific to all known human Wnt ligands. As shown in Fig. 1c, of all the observed 19 Wnt genes, only Wnt3 and Wnt11 mRNA were expressed in the HCC cell line. None of the other known Wnt mRNAs were detected by RT-PCR. After Wnt3 and Wnt11 were identified in the HCC cell line, the mRNA expression level in the HCC cell line was observed by a quantitative real-time RT-PCR (qRT-PCR) assay. Wnt3 mRNA expression level (mean ± SE) in HCC cell lines was 370.0 ± 10.3 for HepG2, 381.3 ± 12.7 for Hep3B, 95.2 ± 6.3 for Huh7, and ^{210.4 ± per 10 9} 18S ribosomal RNAs (18SrRNA). There were 9.5 copies. The expression level of Wnt11 mRNA was 8,499.3 ± 845.0 for HepG2, 290.9 ± 40.1 for Hep3B, and ^{3.57 ± 0.2 copies per 10 9} 18S ribosomal RNA (18SrRNA) in Huh7 cells. Wnt11 mRNA was not detected in focus cells when real-time RT-PCR was used, but this was the same even when conventional RT-PCR was used (FIG. 2B).

Expression of Wnt3, Wnt11 and FZD7 mRNA in human HCC tissue

When the cut-off level was defined as a value of mean ± 3 SD in normal liver tissue, the Wnt3 mRNA expression levels in 13 of 17 HCC tissues (76.5%) and 10 of 17 peri-tumor tissues (58.8%) were It increased compared to the case of the normal control. Compared to normal liver tissue, it was found that the expression level decreased in only one tissue around the tumor. Of the 17 pairs of samples, 12 pairs (70.6%) showed that the intratumoral Wnt3 mRNA expression level was increased compared to the corresponding tissues around the tumor, but the increase was not statistically significant [P = 0.435, Wilcoxon rank sum test] (Fig. 3A). Contrary to the case of Wnt3, it was found that in 11 pairs (64.7%) of the 17 pairs of samples, the level of Wnt11 mRNA expression in the tumor was increased compared to the case of the corresponding tissue around the tumor, but the increase was statistically significant. Not less [P = 0.227, Wilcoxon rank sum test]. However, in 7 of 17 tumor tissues (41.2%), the expression level was lower than the lowest cut-off level of normal liver tissue, whereas it was not in the peri-tumor tissue [P = 0.0036, Fisher's fit test]. In 5 of the 17 tumor tissues (29.4%), the level of Wnt11 mRNA expression was higher than that in the normal liver tissue, but 9 of 17 (52.9%) in the peri-tumor tissues. In both the HCC tissue and the peri-tumor tissue, the FZD7 mRNA expression level was increased in 10 out of 17 samples (58.8%) compared to the normal liver tissue (FIG. 3B ). In 12 of the 17 pairs (70.6%), the level of FZD7 mRNA expression in the tumor increased compared to the corresponding peri-tumor tissue, the difference was statistically significant [P = 0.031, Wilcoxon rank sum test]. (Fig. 3c).

Accumulation of β-catenin in the nucleus

Through immunohistochemical staining, it was found that β-catenin was accumulated in the nucleus in 7 out of 17 HCC tissues (41%). No β-catenin was accumulated in the nucleus of the surrounding tumor progenitor tissue (Fig. 4). In all of the seven samples in which β-catenin was accumulated in the nucleus, β-catenin was also accumulated in the cytoplasm, resulting in a high staining density. Among the 10 tumor tissues in which β-catenin did not accumulate in the nucleus, the density of cytoplasmic staining for β-catenin in one sample was higher than that of the peri-tumor tissue corresponding to the tumor tissue.

By sequencing analysis, mutations occurred in exon 3 of the β-catenin gene in 4 out of 17 HCC samples (23.5%), whereas mutations did not occur in the surrounding tissues around the tumor. And it was found. These mutations were all single missense mutations in codons 35, 37 and 45 (two for I35S, one for S37C, and one for S45F). As a result of staining 3 (42.9%) of 7 samples with β-catenin accumulated in the nucleus by immunohistochemical method, mutations occurred in the sites involved in the phosphorylation and ubiquitination of β-catenin. Could know. One sample in which a mutation occurred in the β-catenin gene (I35S) but did not accumulate in the nucleus was stained with an immunohistochemical method. As a result, the cytoplasm was darkly stained. In all of the remaining 4 samples, where β-catenin was accumulated in the nucleus but no mutations in the β-catenin gene occurred, the FZD7 mRNA level was increased compared to that of the paired pertumoral tissues. FZD7 mRNA levels also increased 7-74 times compared to the average value of normal liver tissue. However, the level of mRNA expression of Wnt3 and/or Wnt11 was not related to whether β-catenin accumulates in the nucleus.

Effects of Wnt3 overexpression in HCC cell lines

In order to measure the effect of Wnt3 on the standardized Wnt pathway in HCC, changes in T-cell factor (Tcf) transcriptional activity were observed after transfection with Wnt3 plasmid. As a result of transfection with the Wnt3 plasmid, both the mRNA expression level (data not shown) and the protein expression level were markedly increased, as shown in FIG. 5A. Tcf transcriptional activity was also increased about 3 times in the case of focus cells compared to the control, which was a statistically significant level (P <0.01). However, Tcf transcriptional activity did not change in Hep3B cells, particularly 24 hours after growth stimulation, and even decreased in Huh7 cells (Fig. 5B). As with these results, the intracellular β-catenin level increased after transfection with the Wnt3 plasmid in the case of focal cells, as evidenced by Western blot analysis, but the intracellular β-catenin level in the Hep3B or Huh7 cells remained unchanged or Or it could be seen that it decreased (FIG. 5A).

Antagonism of Wnt signaling by anti-Wnt3 Ab or Wnt siRNA

Next, the baseline Tcf transcriptional activity and the intracellular β-catenin level were higher in the case of Huh7 and Hep3B cells than in the case of focus cells, so that the effect of anti-Wnt3 Ab or siRNA inhibiting the standardized pathway of the HCC cell line was suppressed. Was observed (Fig. 5). As a result of incubation with polyclonal anti-Wnt3 Ab, luciferase activity decreased by 60% for Huh7, 26% for Hep3B, and 40% for focus cells (Fig. 6A). In order to confirm the inhibitory effect on this standardized pathway, siRNA against Wnt3 was used. First, the inhibition efficiency of three different types of siRNA was measured in Huh7 and Hep3B cells using qRT-PCR assay. As it was seen that the mRNA level was reduced to an average of 50-60% at a concentration of 100 nM, siRNA Wnt3-3 was found to be the most efficient (data not shown) (Fig. 6b). As the mRNA decreased as described above, the Tcf transcriptional activity was also decreased by 48.5% in the case of Huh7, 33% in the case of Hep3B, and 43.5% in the case of focus cells (FIG. 6C). Therefore, it was found that when Wnt3 was inhibited, the standardized pathway of HCC cell lines was also inhibited.

Effect of anti-Wnt3 Ab treatment on wound healing process in HCC cells

Inhibition of Tcf transcriptional activity was also observed as to whether or not the behavior of HCC cells was functionally changed. As a result of performing the wound healing assay using focus cells, it was found that the wound healing process was delayed in the case of cells treated with anti-Wnt3 Ab, and this change was most pronounced after 24 hours. After 24 hours, most of the wounds in focus cells treated with control Ab were covered with migratory cells and/or proliferative cells, whereas this was not the case with anti-Wnt3 Ab-treated cells (FIG. 7).

Through this study, the expression pattern of Wnt ligand in liver and HCC was analyzed. The mRNAs of Wnt3 and Wnt11 were tested using conventional RT-PCR and qRT-PCR methods, indicating that they were expressed in most HCC cell lines. The protein expression level of Wnt11 was also demonstrated using proteomics techniques. Similar to the observations in the HCC cell line, the expression of Wnt3 and Wnt11 mRNA was also confirmed in human liver tissues contained in HCC.

Expression patterns of Wnt3, Wnt11 and FZD7 were observed in human liver tissues, such as HCC tissues and corresponding pertumoral tissues. The mRNA levels of FZD7 in tumor tissue were higher than those in the corresponding pertumoral and normal liver tissues. The difference in the level of Wnt3 mRNA expression between HCC and the peri-tumor tissue was not statistically significant, but in 71% of HCC, the level of Wnt3 expression was increased compared to the corresponding peri-tumor tissue. In addition, in 77% of HCC tissues and 59% of peri-tumor tissues, the Wnt3 mRNA expression level was greater than the "mean ± 3SD" value of normal liver tissue. Through these results, Wnt3 may be upregulated in the early stages of the onset of liver cancer, and/or it is thought that it plays an important role in regenerating liver cells during liver inflammation and liver necrosis.

In addition, in 65% of the paired HCC samples, it was found that the expression level of Wnt11 mRNA in the tumor was decreased compared to the corresponding tissue around the tumor. In 41% of tumor tissues, it was found that the level of Wnt11 expression decreased to below the lowest cut-off level of normal liver tissue. These observations supported the fact that Wnt11 acts as an inhibitor of the standardized pathway.

Judging from the three-fold increase in Tcf transcriptional activity and elevated levels of β-catenin in cells, Wnt3 overexpression was also found to activate the standardized Wnt pathway in focal cells. However, the Tcf transcriptional activity did not change in Hep3B cells, even slightly decreased in Huh7 cells, and the levels of Wnt3 mRNA and protein expression were remarkably elevated after transfection.

When Wnt3 was inhibited by polyclonal anti-Wnt3 Ab or siRNA, Tcf transcriptional activity decreased in all three HCC cell lines. This change was most pronounced in Huh7 cells, which had the highest baseline Tcf transcriptional activity. In addition, treatment of focal cells with anti-Wnt3 Ab inhibited the wound healing process, indicating that this inhibitory effect reduced the migration and proliferation of cells.

In 8 of 17 HCC tissues (47%), β-catenin was accumulated in the nucleus and/or in the cytoplasm, and half of these were mutated in the β-catenin gene. In the remaining four cases where β-catenin was accumulated but no mutation occurred, the level of FZD7 in the tumor was markedly elevated, whereby the upregulation of FZD7 was directly related to the activation of the standardized Wnt signaling pathway in the tumor. Can be seen. The expression level of Wnt3 or Wnt11 was not associated with either β-catenin accumulation or FZD7 accumulation.

In conclusion, Wnt3 and Wnt11 were identified in the HCC cell line and in human liver tissues including the HCC tissue. Wnt3 mRNA expression levels were upregulated in both HCC and peri-tumor tissues compared to normal liver tissue. Wnt11 mRNA expression was downregulated in HCC tissues. Inhibition of Wnt3 by anti-Wnt3 Ab or siRNA interfered with the standardized Wnt signaling pathway and delayed wound healing. However, when Wnt3 was promoted, the Tcf transcriptional activity in focal cells increased. These observations suggest that Wnt3 is a natural Wnt ligand that does not mutate β-catenin during the onset of liver cancer, activates the standardized Wnt signaling pathway, and overexpresses FZD7.

[references]

A number of embodiments of the present invention have been described so far. Nevertheless, it will be understood that the present invention can be further variously modified without departing from the spirit and scope of the present invention. Therefore, other embodiments are also included within the scope of the appended claims below.

SEQUENCE LISTING <110> Rhode Island Hospital, A Lifespan-Partner <120> WNT PROTEINS AND DETECTION AND TREATMENT OF CANCER <130> 18121-003WO2 <140> PCT/US2005/033775 <141> 2005-02-20 <150> US 60/612,098 <151> 2004-09-21 <160> 85 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 574 <212> PRT <213> Homo sapiens <400> 1 Met Arg Asp Pro Gly Ala Ala Val Pro Leu Ser Ser Leu Gly Phe Cys 1 5 10 15 Ala Leu Val Leu Ala Leu Leu Gly Ala Leu Ser Ala Gly Ala Gly Ala 20 25 30 Gln Pro Tyr His Gly Glu Lys Gly Ile Ser Val Pro Asp His Gly Phe 35 40 45 Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 50 55 60 Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly 65 70 75 80 Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 85 90 95 Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105 110 Leu Asp Gln Ala Ile Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Arg 115 120 125 Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Glu 130 135 140 Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu Ile Cys 145 150 155 160 Val Gly Gln Asn Thr Ser Asp Gly Ser Gly Gly Pro Gly Gly Gly Pro 165 170 175 Thr Ala Tyr Pro Thr Ala Pro Tyr Leu Pro Asp Leu Pro Phe Thr Ala 180 185 190 Leu Pro Pro Gly Ala Ser Asp Gly Lys Gly Arg Pro Ala Phe Pro Phe 195 200 205 Ser Cys Pro Arg Gln Leu Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe 210 215 220 Leu Gly Glu Arg Asp Cys Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn 225 230 235 240 Gly Leu Met Tyr Phe Lys Glu Glu Glu Arg Arg Phe Ala Arg Leu Trp 245 250 255 Val Gly Val Trp Ser Val Leu Cys Cys Ala Ser Thr Leu Phe Thr Val 260 265 270 Leu Thr Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro Glu Arg Pro 275 280 285 Ile Ile Phe Leu Ser Gly Cys Tyr Phe Met Val Ala Val Ala His Val 290 295 300 Ala Gly Phe Phe Leu Glu Asp Arg Ala Val Cys Val Glu Arg Phe Ser 305 310 315 320 Asp Asp Gly Tyr Arg Thr Val Ala Gln Gly Thr Lys Lys Glu Gly Cys 325 330 335 Thr Ile Leu Phe Met Val Leu Tyr Phe Phe Gly Met Ala Ser Ser Ile 340 345 350 Trp Trp Val Ile Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys 355 360 365 Trp Gly His Glu Ala Ile Glu Ala Asn Ser Gln Tyr Phe His Leu Ala 370 375 380 Ala Trp Ala Val Pro Ala Val Lys Thr Ile Thr Ile Leu Ala Met Gly 385 390 395 400 Gln Val Asp Gly Asp Leu Leu Asn Gly Val Cys Tyr Val Gly Phe Ser 405 410 415 Ser Val Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe Val Tyr 420 425 430 Phe Phe Ile Gly Thr Ser Phe Leu Leu Ala Gly Phe Val Ser Phe Phe 435 440 445 Arg Ile Arg Thr Ile Met Lys His Asp Gly Thr Lys Thr Glu Lys Leu 450 455 460 Glu Lys Leu Met Val Arg Ile Gly Val Phe Ser Val Leu Tyr Thr Val 465 470 475 480 Pro Ala Thr Ile Val Leu Ala Cys Tyr Phe Tyr Glu Gln Ala Phe Arg 485 490 495 Glu His Trp Glu Arg Thr Trp Leu Leu Gln Thr Cys Lys Ser Tyr Ala 500 505 510 Val Pro Cys Pro Pro Gly His Phe Pro Pro Met Ser Pro Asp Phe Thr 515 520 525 Val Phe Met Ile Lys Cys Leu Met Thr Met Ile Val Gly Ile Thr Thr 530 535 540 Gly Phe Trp Ile Trp Ser Gly Lys Thr Leu Gln Ser Trp Arg Arg Phe 545 550 555 560 Tyr His Arg Leu Ser His Ser Ser Lys Gly Glu Thr Ala Val 565 570 <210> 2 <211> 94 <212> PRT <213> Homo sapiens <400> 2 Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 1 5 10 15 Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly 20 25 30 Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 35 40 45 Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Arg Ser 50 55 60 Leu Cys Glu Arg Ala Arg Gln Gly Cys Glu Ala Leu Met Asn Lys Phe 65 70 75 80 Gly Phe Gln Trp Pro Glu Arg Leu Arg Cys Glu Asn Phe Pro 85 90 <210> 3 <211> 572 <212> PRT <213> Mus musculus <400> 3 Met Arg Gly Pro Gly Thr Ala Ala Ser His Ser Pro Leu Gly Leu Cys 1 5 10 15 Ala Leu Val Leu Ala Leu Leu Gly Ala Leu Pro Thr Asp Thr Arg Ala 20 25 30 Gln Pro Tyr His Gly Glu Lys Gly Ile Ser Val Pro Asp His Gly Phe 35 40 45 Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 50 55 60 Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly 65 70 75 80 Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 85 90 95 Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105 110 Leu Asp Gln Ala Ile Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Arg 115 120 125 Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Glu 130 135 140 Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu Ile Cys 145 150 155 160 Val Gly Gln Asn Thr Ser Asp Gly Ser Gly Gly Ala Gly Gly Ser Pro 165 170 175 Thr Ala Tyr Pro Thr Ala Pro Tyr Leu Pro Asp Pro Pro Phe Thr Ala 180 185 190 Met Ser Pro Ser Asp Gly Arg Gly Arg Leu Ser Phe Pro Phe Ser Cys 195 200 205 Pro Arg Gln Leu Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe Leu Gly 210 215 220 Glu Arg Asp Cys Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn Gly Leu 225 230 235 240 Met Tyr Phe Lys Glu Glu Glu Arg Arg Phe Ala Arg Leu Trp Val Gly 245 250 255 Val Trp Ser Val Leu Ser Cys Ala Ser Thr Leu Phe Thr Val Leu Thr 260 265 270 Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro Glu Arg Pro Ile Ile 275 280 285 Phe Leu Ser Gly Cys Tyr Phe Met Val Ala Val Ala His Val Ala Gly 290 295 300 Phe Leu Leu Glu Asp Arg Ala Val Cys Val Glu Arg Phe Ser Asp Asp 305 310 315 320 Gly Tyr Arg Thr Val Ala Gln Gly Thr Lys Lys Glu Gly Cys Thr Ile 325 330 335 Leu Phe Met Val Leu Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp 340 345 350 Val Ile Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly 355 360 365 His Glu Ala Ile Glu Ala Asn Ser Gln Tyr Phe His Leu Ala Ala Trp 370 375 380 Ala Val Pro Ala Val Lys Thr Ile Thr Ile Leu Ala Met Gly Gln Val 385 390 395 400 Asp Gly Asp Leu Leu Ser Gly Val Cys Tyr Val Gly Leu Ser Ser Val 405 410 415 Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe Val Tyr Leu Phe 420 425 430 Ile Gly Thr Ser Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile 435 440 445 Arg Thr Ile Met Lys His Asp Gly Thr Lys Thr Glu Lys Leu Glu Lys 450 455 460 Leu Met Val Arg Ile Gly Val Phe Ser Val Leu Tyr Thr Val Pro Ala 465 470 475 480 Thr Ile Val Leu Ala Cys Tyr Phe Tyr Glu Gln Ala Phe Arg Glu His 485 490 495 Trp Glu Arg Thr Trp Leu Leu Gln Thr Cys Lys Ser Tyr Ala Val Pro 500 505 510 Cys Pro Pro Arg His Phe Ser Pro Met Ser Pro Asp Phe Thr Val Phe 515 520 525 Met Ile Lys Tyr Leu Met Thr Met Ile Val Gly Ile Thr Thr Gly Phe 530 535 540 Trp Ile Trp Ser Gly Lys Thr Leu Gln Ser Trp Arg Arg Phe Tyr His 545 550 555 560 Arg Leu Ser His Ser Ser Lys Gly Glu Thr Ala Val 565 570 <210> 4 <211> 694 <212> PRT <213> Homo sapiens <400> 4 Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu 1 5 10 15 Ala Leu Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys Glu 20 25 30 Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly Ile Gly Tyr 35 40 45 Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His Asp Thr Gln Asp Glu 50 55 60 Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu Val Glu Ile Gln Cys 65 70 75 80 Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95 Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 105 110 Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125 Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135 140 Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr Ala Ala 145 150 155 160 Pro Ser Pro Pro Arg Arg Leu Pro Pro Pro Pro Pro Gly Glu Gln Pro 165 170 175 Pro Ser Gly Ser Gly His Gly Arg Pro Pro Gly Ala Arg Pro Pro His 180 185 190 Arg Gly Gly Gly Arg Gly Gly Gly Gly Gly Asp Ala Ala Ala Pro Pro 195 200 205 Ala Arg Gly Gly Gly Gly Gly Gly Lys Ala Arg Pro Pro Gly Gly Gly 210 215 220 Ala Ala Pro Cys Glu Pro Gly Cys Gln Cys Arg Ala Pro Met Val Ser 225 230 235 240 Val Ser Ser Glu Arg His Pro Leu Tyr Asn Arg Val Lys Thr Gly Gln 245 250 255 Ile Ala Asn Cys Ala Leu Pro Cys His Asn Pro Phe Phe Ser Gln Asp 260 265 270 Glu Arg Ala Phe Thr Val Phe Trp Ile Gly Leu Trp Ser Val Leu Cys 275 280 285 Phe Val Ser Thr Phe Ala Thr Val Ser Thr Phe Leu Ile Asp Met Glu 290 295 300 Arg Phe Lys Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser Ala Cys Tyr 305 310 315 320 Leu Phe Val Ser Val Gly Tyr Leu Val Arg Leu Val Ala Gly His Glu 325 330 335 Lys Val Ala Cys Ser Gly Gly Ala Pro Gly Ala Gly Gly Ala Gly Gly 340 345 350 Ala Gly Gly Ala Ala Ala Gly Ala Gly Ala Ala Gly Ala Gly Ala Gly 355 360 365 Gly Pro Gly Gly Arg Gly Glu Tyr Glu Glu Leu Gly Ala Val Glu Gln 370 375 380 His Val Arg Tyr Glu Thr Thr Gly Pro Ala Leu Cys Thr Val Val Phe 385 390 395 400 Leu Leu Val Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp Val Ile 405 410 415 Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly Asn Glu 420 425 430 Ala Ile Ala Gly Tyr Ser Gln Tyr Phe His Leu Ala Ala Trp Leu Val 435 440 445 Pro Ser Val Lys Ser Ile Ala Val Leu Ala Leu Ser Ser Val Asp Gly 450 455 460 Asp Pro Val Ala Gly Ile Cys Tyr Val Gly Asn Gln Ser Leu Asp Asn 465 470 475 480 Leu Arg Gly Phe Val Leu Ala Pro Leu Val Ile Tyr Leu Phe Ile Gly 485 490 495 Thr Met Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser 500 505 510 Val Ile Lys Gln Gln Asp Gly Pro Thr Lys Thr His Lys Leu Glu Lys 515 520 525 Leu Met Ile Arg Leu Gly Leu Phe Thr Val Leu Tyr Thr Val Pro Ala 530 535 540 Ala Val Val Val Ala Cys Leu Phe Tyr Glu Gln His Asn Arg Pro Arg 545 550 555 560 Trp Glu Ala Thr His Asn Cys Pro Cys Leu Arg Asp Leu Gln Pro Asp 565 570 575 Gln Ala Arg Arg Pro Asp Tyr Ala Val Phe Met Leu Lys Tyr Phe Met 580 585 590 Cys Leu Val Val Gly Ile Thr Ser Gly Val Trp Val Trp Ser Gly Lys 595 600 605 Thr Leu Glu Ser Trp Arg Ser Leu Cys Thr Arg Cys Cys Trp Ala Ser 610 615 620 Lys Gly Ala Ala Val Gly Gly Gly Ala Gly Ala Thr Ala Ala Gly Gly 625 630 635 640 Gly Gly Gly Pro Gly Gly Gly Gly Gly Gly Gly Pro Gly Gly Gly Gly 645 650 655 Gly Pro Gly Gly Gly Gly Gly Ser Leu Tyr Ser Asp Val Ser Thr Gly 660 665 670 Leu Thr Trp Arg Ser Gly Thr Ala Ser Ser Ser Val Ser Tyr Pro Lys Gln 675 680 685 Met Pro Leu Ser Gln Val 690 <210> 5 <211> 105 <212> PRT <213> Homo sapiens <400> 5 Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly Ile Gly Tyr Asn Tyr 1 5 10 15 Thr Tyr Met Pro Asn Gln Phe Asn His Asp Thr Gln Asp Glu Ala Gly 20 25 30 Leu Glu Val His Gln Phe Trp Pro Leu Val Glu Ile Gln Cys Ser Pro 35 40 45 Asp Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys Leu Glu 50 55 60 Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 65 70 75 80 Lys Ala Gly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe Ala Trp Pro 85 90 95 Asp Arg Met Arg Cys Asp Arg Leu Pro 100 105 <210> 6 <211> 685 <212> PRT <213> Mus musculus <400> 6 Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu 1 5 10 15 Ala Val Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys Glu 20 25 30 Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly Ile Gly Tyr 35 40 45 Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His Asp Thr Gln Asp Glu 50 55 60 Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu Val Glu Ile Gln Cys 65 70 75 80 Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95 Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 105 110 Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125 Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135 140 Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr Ala Ala 145 150 155 160 Pro Ser Pro Pro Arg Arg Leu Pro Pro Pro Pro Pro Pro Gly Glu Gln 165 170 175 Pro Pro Ser Gly Ser Gly His Ser Arg Pro Pro Gly Ala Arg Pro Pro 180 185 190 His Arg Gly Gly Ser Ser Arg Gly Ser Gly Asp Ala Ala Ala Ala Pro 195 200 205 Pro Ser Arg Gly Gly Lys Ala Arg Pro Pro Gly Gly Gly Ala Ala Pro 210 215 220 Cys Glu Pro Gly Cys Gln Cys Arg Ala Pro Met Val Ser Val Ser Ser 225 230 235 240 Glu Arg His Pro Leu Tyr Asn Arg Val Lys Thr Gly Gln Ile Ala Asn 245 250 255 Cys Ala Leu Pro Cys His Asn Pro Phe Phe Ser Gln Asp Glu Arg Ala 260 265 270 Phe Thr Val Phe Trp Ile Gly Leu Trp Ser Val Leu Cys Phe Val Ser 275 280 285 Thr Phe Ala Thr Val Ser Thr Phe Leu Ile Asp Met Glu Arg Phe Lys 290 295 300 Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser Ala Cys Tyr Leu Phe Val 305 310 315 320 Ser Val Gly Tyr Leu Val Arg Leu Val Ala Gly His Glu Lys Val Ala 325 330 335 Cys Ser Gly Gly Ala Pro Gly Ala Gly Gly Arg Gly Gly Ala Gly Gly 340 345 350 Ala Ala Ala Ala Gly Ala Gly Ala Ala Gly Arg Gly Ala Ser Ser Pro 355 360 365 Gly Ala Arg Gly Glu Tyr Glu Glu Leu Gly Ala Val Glu Gln His Val 370 375 380 Arg Tyr Glu Thr Thr Gly Pro Ala Leu Cys Thr Val Val Phe Leu Leu 385 390 395 400 Val Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp Val Ile Leu Ser 405 410 415 Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly Asn Glu Ala Ile 420 425 430 Ala Gly Tyr Ser Gln Tyr Phe His Leu Ala Ala Trp Leu Val Pro Ser 435 440 445 Val Lys Ser Ile Ala Val Leu Ala Leu Ser Ser Val Asp Gly Asp Pro 450 455 460 Val Ala Gly Ile Cys Tyr Val Gly Asn Gln Ser Leu Asp Asn Leu Arg 465 470 475 480 Gly Phe Val Leu Ala Pro Leu Val Ile Tyr Leu Phe Ile Gly Thr Met 485 490 495 Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser Val Ile 500 505 510 Lys Gln Gln Gly Gly Pro Thr Lys Thr His Lys Leu Glu Lys Leu Met 515 520 525 Ile Arg Leu Gly Leu Phe Thr Val Leu Tyr Thr Val Pro Ala Ala Val 530 535 540 Val Val Ala Cys Leu Phe Tyr Glu Gln His Asn Arg Pro Arg Trp Glu 545 550 555 560 Ala Thr His Asn Cys Pro Cys Leu Arg Asp Leu Gln Pro Asp Gln Ala 565 570 575 Arg Arg Pro Asp Tyr Ala Val Phe Met Leu Lys Tyr Phe Met Cys Leu 580 585 590 Val Val Gly Ile Thr Ser Gly Val Trp Val Trp Ser Gly Lys Thr Leu 595 600 605 Glu Ser Trp Arg Ala Leu Cys Thr Arg Cys Cys Trp Ala Ser Lys Gly 610 615 620 Ala Ala Val Gly Ala Gly Ala Gly Gly Ser Gly Pro Gly Gly Ser Gly 625 630 635 640 Pro Gly Pro Gly Gly Gly Gly Gly His Gly Gly Gly Gly Gly Ser Leu 645 650 655 Tyr Ser Asp Val Ser Thr Gly Leu Thr Trp Arg Ser Gly Thr Ala Ser 660 665 670 Ser Val Ser Tyr Pro Lys Gln Met Pro Leu Ser Gln Val 675 680 685 <210> 7 <211> 355 <212> PRT <213> Homo sapiens <400> 7 Met Glu Pro His Leu Leu Gly Leu Leu Leu Gly Leu Leu Leu Gly Gly 1 5 10 15 Thr Arg Val Leu Ala Gly Tyr Pro Ile Trp Trp Ser Leu Ala Leu Gly 20 25 30 Gln Gln Tyr Thr Ser Leu Gly Ser Gln Pro Leu Leu Cys Gly Ser Ile 35 40 45 Pro Gly Leu Val Pro Lys Gln Leu Arg Phe Cys Arg Asn Tyr Ile Glu 50 55 60 Ile Met Pro Ser Val Ala Glu Gly Val Lys Leu Gly Ile Gln Glu Cys 65 70 75 80 Gln His Gln Phe Arg Gly Arg Arg Trp Asn Cys Thr Thr Ile Asp Asp 85 90 95 Ser Leu Ala Ile Phe Gly Pro Val Leu Asp Lys Ala Thr Arg Glu Ser 100 105 110 Ala Phe Val His Ala Ile Ala Ser Ala Gly Val Ala Phe Ala Val Thr 115 120 125 Arg Ser Cys Ala Glu Gly Thr Ser Thr Ile Cys Gly Cys Asp Ser His 130 135 140 His Lys Gly Pro Pro Gly Glu Gly Trp Lys Trp Gly Gly Cys Ser Glu 145 150 155 160 Asp Ala Asp Phe Gly Val Leu Val Ser Arg Glu Phe Ala Asp Ala Arg 165 170 175 Glu Asn Arg Pro Asp Ala Arg Ser Ala Met Asn Lys His Asn Asn Glu 180 185 190 Ala Gly Arg Thr Thr Ile Leu Asp His Met His Leu Lys Cys Lys Cys 195 200 205 His Gly Leu Ser Gly Ser Cys Glu Val Lys Thr Cys Trp Trp Ala Gln 210 215 220 Pro Asp Phe Arg Ala Ile Gly Asp Phe Leu Lys Asp Lys Tyr Asp Ser 225 230 235 240 Ala Ser Glu Met Val Val Glu Lys His Arg Glu Ser Arg Gly Trp Val 245 250 255 Glu Thr Leu Arg Ala Lys Tyr Ser Leu Phe Lys Pro Pro Thr Glu Arg 260 265 270 Asp Leu Val Tyr Tyr Glu Asn Ser Pro Asn Phe Cys Glu Pro Asn Pro 275 280 285 Glu Thr Gly Ser Phe Gly Thr Arg Asp Arg Thr Cys Asn Val Thr Ser 290 295 300 His Gly Ile Asp Gly Cys Asp Leu Leu Cys Cys Gly Arg Gly His Asn 305 310 315 320 Thr Arg Thr Glu Lys Arg Lys Glu Lys Cys His Cys Ile Phe His Trp 325 330 335 Cys Cys Tyr Val Ser Cys Gln Glu Cys Ile Arg Ile Tyr Asp Val His 340 345 350 Thr Cys Lys 355 <210> 8 <211> 15 <212> PRT <213> Homo sapiens <400> 8 Arg Glu Ser Ala Phe Val His Ala Ile Ala Ser Ala Gly Val Ala 1 5 10 15 <210> 9 <211> 14 <212> PRT <213> Homo sapiens <400> 9 Arg Ser Cys Ala Glu Gly Thr Ser Thr Ile Cys Gly Cys Asp 1 5 10 <210> 10 <211> 13 <212> PRT <213> Homo sapiens <400> 10 Trp Lys Trp Gly Gly Cys Ser Glu Asp Ala Asp Phe Gly 1 5 10 <210> 11 <211> 16 <212> PRT <213> Homo sapiens <400> 11 Cys Lys Cys His Gly Leu Ser Gly Ser Cys Glu Val Lys Thr Cys Trp 1 5 10 15 <210> 12 <211> 12 <212> PRT <213> Homo sapiens <400> 12 Asp Leu Val Tyr Tyr Glu Asn Ser Pro Asn Phe Cys 1 5 10 <210> 13 <211> 355 <212> PRT <213> Mus musculus <400> 13 Met Glu Pro His Leu Leu Gly Leu Leu Leu Gly Leu Leu Leu Ser Gly 1 5 10 15 Thr Arg Val Leu Ala Gly Tyr Pro Ile Trp Trp Ser Leu Ala Leu Gly 20 25 30 Gln Gln Tyr Thr Ser Leu Ala Ser Gln Pro Leu Leu Cys Gly Ser Ile 35 40 45 Pro Gly Leu Val Pro Lys Gln Leu Arg Phe Cys Arg Asn Tyr Ile Glu 50 55 60 Ile Met Pro Ser Val Ala Glu Gly Val Lys Leu Gly Ile Gln Glu Cys 65 70 75 80 Gln His Gln Phe Arg Gly Arg Arg Trp Asn Cys Thr Thr Ile Asp Asp 85 90 95 Ser Leu Ala Ile Phe Gly Pro Val Leu Asp Lys Ala Thr Arg Glu Ser 100 105 110 Ala Phe Val His Ala Ile Ala Ser Ala Gly Val Ala Phe Ala Val Thr 115 120 125 Arg Ser Cys Ala Glu Gly Thr Ser Thr Ile Cys Gly Cys Asp Ser His 130 135 140 His Lys Gly Pro Pro Gly Glu Gly Trp Lys Trp Gly Gly Cys Ser Glu 145 150 155 160 Asp Ala Asp Phe Gly Val Leu Val Ser Arg Glu Phe Ala Asp Ala Arg 165 170 175 Glu Asn Arg Pro Asp Ala Arg Ser Ala Met Asn Lys His Asn Asn Glu 180 185 190 Ala Gly Arg Thr Thr Ile Leu Asp His Met His Leu Lys Cys Lys Cys 195 200 205 His Gly Leu Ser Gly Ser Cys Glu Val Lys Thr Cys Trp Trp Ala Gln 210 215 220 Pro Asp Phe Arg Ala Ile Gly Asp Phe Leu Lys Asp Lys Tyr Asp Ser 225 230 235 240 Ala Ser Glu Met Val Val Glu Lys His Arg Glu Ser Arg Gly Trp Val 245 250 255 Glu Thr Leu Arg Ala Lys Tyr Ala Leu Phe Lys Pro Pro Thr Glu Arg 260 265 270 Asp Leu Val Tyr Tyr Glu Asn Ser Pro Asn Phe Cys Glu Pro Asn Pro 275 280 285 Glu Thr Gly Ser Phe Gly Thr Arg Asp Arg Thr Cys Asn Val Thr Ser 290 295 300 His Gly Ile Asp Gly Cys Asp Leu Leu Cys Cys Gly Arg Gly His Asn 305 310 315 320 Thr Arg Thr Glu Lys Arg Lys Glu Lys Cys His Cys Val Phe His Trp 325 330 335 Cys Cys Tyr Val Ser Cys Gln Glu Cys Ile Arg Ile Tyr Asp Val His 340 345 350 Thr Cys Lys 355 <210> 14 <211> 351 <212> PRT <213> Homo sapiens <400> 14 Met Phe Leu Ser Lys Pro Ser Val Tyr Ile Cys Leu Phe Thr Cys Val 1 5 10 15 Leu Gln Leu Ser His Ser Trp Ser Val Asn Asn Phe Leu Met Thr Gly 20 25 30 Pro Lys Ala Tyr Leu Ile Tyr Ser Ser Ser Val Ala Ala Gly Ala Gln 35 40 45 Ser Gly Ile Glu Glu Cys Lys Tyr Gln Phe Ala Trp Asp Arg Trp Asn 50 55 60 Cys Pro Glu Arg Ala Leu Gln Leu Ser Ser His Gly Gly Leu Arg Ser 65 70 75 80 Ala Asn Arg Glu Thr Ala Phe Val His Ala Ile Ser Ser Ala Gly Val 85 90 95 Met Tyr Thr Leu Thr Arg Asn Cys Ser Leu Gly Asp Phe Asp Asn Cys 100 105 110 Gly Cys Asp Asp Ser Arg Asn Gly Gln Leu Gly Gly Gln Gly Trp Leu 115 120 125 Trp Gly Gly Cys Ser Asp Asn Val Gly Phe Gly Glu Ala Ile Ser Lys 130 135 140 Gln Phe Val Asp Ala Leu Glu Thr Gly Gln Asp Ala Arg Ala Ala Met 145 150 155 160 Asn Leu His Asn Asn Glu Ala Gly Arg Lys Ala Val Lys Gly Thr Met 165 170 175 Lys Arg Thr Cys Lys Cys His Gly Val Ser Gly Ser Cys Thr Thr Gln 180 185 190 Thr Cys Trp Leu Gln Leu Pro Glu Phe Arg Glu Val Gly Ala His Leu 195 200 205 Lys Glu Lys Tyr His Ala Ala Leu Lys Val Asp Leu Leu Gln Gly Ala 210 215 220 Gly Asn Ser Ala Ala Ala Arg Gly Ala Ile Ala Asp Thr Phe Arg Ser 225 230 235 240 Ile Ser Thr Arg Glu Leu Val His Leu Glu Asp Ser Pro Asp Tyr Cys 245 250 255 Leu Glu Asn Lys Thr Leu Gly Leu Leu Gly Thr Glu Gly Arg Glu Cys 260 265 270 Leu Arg Arg Gly Arg Ala Leu Gly Arg Trp Glu Leu Arg Ser Cys Arg 275 280 285 Arg Leu Cys Gly Asp Cys Gly Leu Ala Val Glu Glu Arg Arg Ala Glu 290 295 300 Thr Val Ser Ser Cys Asn Cys Lys Phe His Trp Cys Cys Ala Val Arg 305 310 315 320 Cys Glu Gln Cys Arg Arg Arg Val Thr Lys Tyr Phe Cys Ser Arg Ala 325 330 335 Glu Arg Pro Arg Gly Gly Ala Ala His Lys Pro Gly Arg Lys Pro 340 345 350 <210> 15 <211> 15 <212> PRT <213> Homo sapiens <400> 15 Arg Glu Thr Ala Phe Val His Ala Ile Ser Ser Ala Gly Val Met 1 5 10 15 <210> 16 <211> 14 <212> PRT <213> Homo sapiens <400> 16 Arg Asn Cys Ser Leu Gly Asp Phe Asp Asn Cys Gly Cys Asp 1 5 10 <210> 17 <211> 13 <212> PRT <213> Homo sapiens <400> 17 Trp Leu Trp Gly Gly Cys Ser Asp Asn Val Gly Phe Gly 1 5 10 <210> 18 <211> 16 <212> PRT <213> Homo sapiens <400> 18 Cys Lys Cys His Gly Val Ser Gly Ser Cys Thr Thr Gln Thr Cys Trp 1 5 10 15 <210> 19 <211> 12 <212> PRT <213> Homo sapiens <400> 19 Glu Leu Val His Leu Glu Asp Ser Pro Asp Tyr Cys 1 5 10 <210> 20 <211> 350 <212> PRT <213> Mus musculus <400> 20 Met Phe Leu Met Lys Pro Val Cys Val Leu Leu Val Thr Cys Val Leu 1 5 10 15 His Arg Ser His Ala Trp Ser Val Asn Asn Phe Leu Met Thr Gly Pro 20 25 30 Lys Ala Tyr Leu Val Tyr Ser Ser Ser Val Ala Ala Gly Ala Gln Ser 35 40 45 Gly Ile Glu Glu Cys Lys Tyr Gln Phe Ala Trp Asp Arg Trp Asn Cys 50 55 60 Pro Glu Arg Ala Leu Gln Leu Ser Ser His Gly Gly Leu Arg Ser Ala 65 70 75 80 Asn Arg Glu Thr Ala Phe Val His Ala Ile Ser Ser Ala Gly Val Met 85 90 95 Tyr Thr Leu Thr Arg Asn Cys Ser Leu Gly Asp Phe Asp Asn Cys Gly 100 105 110 Cys Asp Asp Ser Arg Asn Gly Gln Leu Gly Gly Gln Gly Trp Leu Trp 115 120 125 Gly Gly Cys Ser Asp Asn Val Gly Phe Gly Glu Ala Ile Ser Lys Gln 130 135 140 Phe Val Asp Ala Leu Glu Thr Gly Gln Asp Ala Arg Ala Ala Met Asn 145 150 155 160 Leu His Asn Asn Glu Ala Gly Arg Lys Ala Val Lys Gly Thr Met Lys 165 170 175 Arg Thr Cys Lys Cys His Gly Val Ser Gly Ser Cys Thr Thr Gln Thr 180 185 190 Cys Trp Leu Gln Leu Pro Glu Phe Arg Glu Val Gly Ala His Leu Lys 195 200 205 Glu Lys Tyr His Ala Ala Leu Lys Val Asp Leu Leu Gln Gly Ala Gly 210 215 220 Asn Ser Ala Ala Gly Arg Gly Ala Ile Ala Asp Thr Phe Arg Ser Ile 225 230 235 240 Ser Thr Arg Glu Leu Val His Leu Glu Asp Ser Pro Asp Tyr Cys Leu 245 250 255 Glu Asn Lys Thr Leu Gly Leu Leu Gly Thr Glu Gly Arg Glu Cys Leu 260 265 270 Arg Arg Gly Arg Ala Leu Gly Arg Trp Glu Arg Arg Ser Cys Arg Arg 275 280 285 Leu Cys Gly Asp Cys Gly Leu Ala Val Glu Glu Arg Arg Ala Glu Thr 290 295 300 Val Ser Ser Cys Asn Cys Lys Phe His Trp Cys Cys Ala Val Arg Cys 305 310 315 320 Glu Gln Cys Arg Arg Arg Val Thr Lys Tyr Phe Cys Ser Arg Ala Glu 325 330 335 Arg Pro Pro Arg Gly Ala Ala His Lys Pro Gly Lys Asn Ser 340 345 350 <210> 21 <211> 354 <212> PRT <213> Homo sapiens <400> 21 Met Arg Ala Arg Pro Gln Val Cys Glu Ala Leu Leu Phe Ala Leu Ala 1 5 10 15 Leu Gln Thr Gly Val Cys Tyr Gly Ile Lys Trp Leu Ala Leu Ser Lys 20 25 30 Thr Pro Ser Ala Leu Ala Leu Asn Gln Thr Gln His Cys Lys Gln Leu 35 40 45 Glu Gly Leu Val Ser Ala Gln Val Gln Leu Cys Arg Ser Asn Leu Glu 50 55 60 Leu Met His Thr Val Val His Ala Ala Arg Glu Val Met Lys Ala Cys 65 70 75 80 Arg Arg Ala Phe Ala Asp Met Arg Trp Asn Cys Ser Ser Ile Glu Leu 85 90 95 Ala Pro Asn Tyr Leu Leu Asp Leu Glu Arg Gly Thr Arg Glu Ser Ala 100 105 110 Phe Val Tyr Ala Leu Ser Ala Ala Ala Ile Ser His Ala Ile Ala Arg 115 120 125 Ala Cys Thr Ser Gly Asp Leu Pro Gly Cys Ser Cys Gly Pro Val Pro 130 135 140 Gly Glu Pro Pro Gly Pro Gly Asn Arg Trp Gly Gly Cys Ala Asp Asn 145 150 155 160 Leu Ser Tyr Gly Leu Leu Met Gly Ala Lys Phe Ser Asp Ala Pro Met 165 170 175 Lys Val Lys Lys Thr Gly Ser Gln Ala Asn Lys Leu Met Arg Leu His 180 185 190 Asn Ser Glu Val Gly Arg Gln Ala Leu Arg Ala Ser Leu Glu Met Lys 195 200 205 Cys Lys Cys His Gly Val Ser Gly Ser Cys Ser Ile Arg Thr Cys Trp 210 215 220 Lys Gly Leu Gln Glu Leu Gln Asp Val Ala Ala Asp Leu Lys Thr Arg 225 230 235 240 Tyr Leu Ser Ala Thr Lys Val Val His Arg Pro Met Gly Thr Arg Lys 245 250 255 His Leu Val Pro Lys Asp Leu Asp Ile Arg Pro Val Lys Asp Ser Glu 260 265 270 Leu Val Tyr Leu Gln Ser Ser Pro Asp Phe Cys Met Lys Asn Glu Lys 275 280 285 Val Gly Ser His Gly Thr Gln Asp Arg Gln Cys Asn Lys Thr Ser Asn 290 295 300 Gly Ser Asp Ser Cys Asp Leu Met Cys Cys Gly Arg Gly Tyr Asn Pro 305 310 315 320 Tyr Thr Asp Arg Val Val Glu Arg Cys His Cys Lys Tyr His Trp Cys 325 330 335 Cys Tyr Val Thr Cys Arg Arg Cys Glu Arg Thr Val Glu Arg Tyr Val 340 345 350 Cys Lys <210> 22 <211> 15 <212> PRT <213> Homo sapiens <400> 22 Arg Glu Ser Ala Phe Val Tyr Ala Leu Ser Ala Ala Ala Ile Ser 1 5 10 15 <210> 23 <211> 14 <212> PRT <213> Homo sapiens <400> 23 Arg Ala Cys Thr Ser Gly Asp Leu Pro Gly Cys Ser Cys Gly 1 5 10 <210> 24 <211> 13 <212> PRT <213> Homo sapiens <400> 24 Asn Arg Trp Gly Gly Cys Ala Asp Asn Leu Ser Tyr Gly 1 5 10 <210> 25 <211> 16 <212> PRT <213> Homo sapiens <400> 25 Cys Lys Cys His Gly Val Ser Gly Ser Cys Ser Ile Arg Thr Cys Trp 1 5 10 15 <210> 26 <211> 12 <212> PRT <213> Homo sapiens <400> 26 Glu Leu Val Tyr Leu Gln Ser Ser Pro Asp Phe Cys 1 5 10 <210> 27 <211> 354 <212> PRT <213> Mus musculus <400> 27 Met Arg Ala Arg Pro Gln Val Cys Glu Ala Leu Leu Phe Ala Leu Ala 1 5 10 15 Leu His Thr Gly Val Cys Tyr Gly Ile Lys Trp Leu Ala Leu Ser Lys 20 25 30 Thr Pro Ala Ala Leu Ala Leu Asn Gln Thr Gln His Cys Lys Gln Leu 35 40 45 Glu Gly Leu Val Ser Ala Gln Val Gln Leu Cys Arg Ser Asn Leu Glu 50 55 60 Leu Met Arg Thr Ile Val His Ala Ala Arg Gly Ala Met Lys Ala Cys 65 70 75 80 Arg Arg Ala Phe Ala Asp Met Arg Trp Asn Cys Ser Ser Ile Glu Leu 85 90 95 Ala Pro Asn Tyr Leu Leu Asp Leu Glu Arg Gly Thr Arg Glu Ser Ala 100 105 110 Phe Val Tyr Ala Leu Ser Ala Ala Thr Ile Ser His Thr Ile Ala Arg 115 120 125 Ala Cys Thr Ser Gly Asp Leu Pro Gly Cys Ser Cys Gly Pro Val Pro 130 135 140 Gly Glu Pro Pro Gly Pro Gly Asn Arg Trp Gly Gly Cys Ala Asp Asn 145 150 155 160 Leu Ser Tyr Gly Leu Leu Met Gly Ala Lys Phe Ser Asp Ala Pro Met 165 170 175 Lys Val Lys Lys Thr Gly Ser Gln Ala Asn Lys Leu Met Arg Leu His 180 185 190 Asn Ser Glu Val Gly Arg Gln Ala Leu Arg Ala Ser Leu Glu Thr Lys 195 200 205 Cys Lys Cys His Gly Val Ser Gly Ser Cys Ser Ile Arg Thr Cys Trp 210 215 220 Lys Gly Leu Gln Glu Leu Gln Asp Val Ala Ala Asp Leu Lys Thr Arg 225 230 235 240 Tyr Leu Ser Ala Thr Lys Val Val His Arg Pro Met Gly Thr Arg Lys 245 250 255 His Leu Val Pro Lys Asp Leu Asp Ile Arg Pro Val Lys Asp Ser Glu 260 265 270 Leu Val Tyr Leu Gln Ser Ser Pro Asp Phe Cys Met Lys Asn Glu Lys 275 280 285 Val Gly Ser His Gly Thr Gln Asp Arg Gln Cys Asn Lys Thr Ser Asn 290 295 300 Gly Ser Asp Ser Cys Asp Leu Met Cys Cys Gly Arg Gly Tyr Asn Pro 305 310 315 320 Tyr Thr Asp Arg Val Val Glu Arg Cys His Cys Lys Tyr His Trp Cys 325 330 335 Cys Tyr Val Thr Cys Arg Arg Cys Glu Arg Thr Val Glu Arg Tyr Val 340 345 350 Cys Lys <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 28 gaaatcccat caccatcttc cag 23 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 29 atgagtcctt ccacgatacc aaag 24 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 30 tgttgcctgg ctgggtttc 19 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 31 ctgtaagcag gttcgtggag 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 32 gtggatgcaa aggaaaggaa 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 33 agccagcatg tcctgagagt 20 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 34 acccaagatg gtgccaactt c 21 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 35 cacaaccgtc tgttcctttt gatg 24 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 36 ggagtgtatt cgcatctacg acg 23 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 37 cgagttgggt ctgggtcatt tac 23 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 38 ccccactcgg atacttctta ctcc 24 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 39 ctcctggatg ccaatcttga tg 22 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 40 tttgtggatg tgcgggagag 20 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 41 atctgtgtgc ggcttgaact g 21 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 42 acacctcttt ccaaacaggc c 21 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 43 ggattgttaa actcaactct c 21 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 44 ggagcgagag aagaactttg cc 22 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 45 gaagcagcac cagtggaact tg 22 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 46 cttggttatg gaccctaccc aggcatc 27 <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 47 cactgcagca gctcgcccat agaa 24 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 48 gctgcctggg ccacctcttt ctca 24 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 49 cccggtggta caggccttgc ttct 24 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 50 tcaacgagtg ccagtaccag 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 51 ccctcggctt ggttgtagta 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 52 tccagtttgc ttgggaacgc 20 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 53 ccatcacagc cacagttttc g 21 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 54 catctgtctt ttcacctgtg tcctc 25 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 55 aatgctgtct cccgattggc 20 <210> 56 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 56 tctgggtgct cctgttcttc ctac 24 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 57 attggtgttg gcattcgtgg 20 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 58 actgtcccga ggcaagagtt tc 22 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 59 gcatttccgc ttcaggtttt c 21 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 60 tgctgacctc aagacccgat ac 22 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 61 tgtcgcttcc gttggatgtc 20 <210> 62 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 62 tgccagttcc agttccgctt tg 22 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 63 ttcacaccca cgaggttgtt g 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 64 tgagtgccag tttcagttcc g 21 <210> 65 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 65 cttgtttcct ctcttggacc cc 22 <210> 66 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 66 ctgctccgat gatgtccagt atg 23 <210> 67 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 67 cattctctgc cttgtgtccc tg 22 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 68 acttcggcgt gttagtgtcc 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 69 catttgaggt gcatgtggtc 20 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 70 ttccgatgct cctatgaagg 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 71 agacacccca tggcacttac 20 <210> 72 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 72 gccgcttcta ccacagact 19 <210> 73 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 73 ttcataccgc agtctcccc 19 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 74 ggacacggac aggattgaca 20 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 75 acccacggaa tcgagaaaga 20 <210> 76 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 76 tagttaagct taccatggag ccccacctgc tc 32 <210> 77 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 77 gcagctgacg tagcagcacc agtggaagat gcagtg 36 <210> 78 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 78 gtagatgcga atacactcct ggcagctgac gtagca 36 <210> 79 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 79 tgcttgaatt ccttgcaggt gtgcacgtcg tagatgcgaa taca 44 <210> 80 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetically generated peptide <400> 80 Leu Arg Ala Lys Tyr Ser Leu Phe Lys Pro Pro Thr Glu Arg Asp Leu 1 5 10 15 <210> 81 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Synthetically generated siRNA <400> 81 ggaaaaaugc cacugcauc 19 <210> 82 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Synthetically generated siRNA <400> 82 ggaguguauu cgcaucuac 19 <210> 83 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Synthetically generated siRNA <400> 83 ggcuuaucuu ugcacaugu 19 <210> 84 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 84 gatttgatgg agttggacat gg 22 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 85 tgttcttgag tgaaggactg ag 22

Claims (1)

As a method of identifying anticancer drugs,
Selecting a test compound that binds to a polypeptide comprising the amino acid sequence of a Wnt3, Wnt8b or Wnt11 protein or FZD-binding fragment thereof; And
This test compound
(i) can reduce Wnt / FZD7 signaling in cells;
(ii) can reduce liver cancer cell mobility;
(iii) reduce the accumulation of β-catenin in liver cancer cells; or
(iv) whether liver cancer can be treated in vitro or in vivo;
To measure
Wherein the test compound that retains the efficacy of at least one of (i)-(iv) is an anticancer agent.

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