Antibody for Recognizing Specific Motif of WLS Protein, and Pharmaceutical Composition Comprising Same

Abstract

The present invention relates to an antibody that recognizes a specific motif of WLS protein so as to inhibit overactivation of the Wnt signaling pathway to thereby prevent or treat a disease associated with the Wnt signaling pathway, and to a pharmaceutical composition containing the same.

Claims

1. A method for preventing or treating a Wnt signaling pathway-associated disease, comprising administering to a subject in need of such treatment with an effective amount of (a) an antibody specific for a protein represented by an amino acid sequence selected from the group consisting of: (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3; (ii) an amino acid sequence comprising at least 6 consecutive amino acids of the amino acid sequence of any one of SEQ ID NOs: 1 to 3; and (iii) an amino acid sequence having a homology of at least 90% to the amino acid sequence of (i) or (ii), or (b) one or more selected from the group consisting of antisense oligonucleotide, siRNA, shRNA and microRNA, which are specific for a gene encoding an amino acid sequence selected from the group consisting of: (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3; (ii) an amino acid sequence comprising at least 6 consecutive amino acids of the amino acid sequence of any one of SEQ ID NOs: 1 to 3; and (iii) an amino acid sequence having a homology of at least 90% to the amino acid sequence of (i) or (ii).

2. The method of claim 1, wherein the protein is represented by SEQ ID NO: 2.

3. The method of claim 1, wherein the protein is represented by any one of SEQ ID NOs: 4 to 6.

4. The method of claim 1, wherein the protein is represented by SEQ ID NO: 6.

5. The method of claim 1, wherein the Wnt signaling pathway-associated disease is selected from the group consisting of cancer, retinopathy, macular degeneration, fibrosis, fungal or viral infection, bone or cartilage disease, osteoarthritis, rheumatoid arthritis, neurodegenerative disease, diabetes, a disease of the digestive system, cardiovascular disease and renal disease.

6. The method of claim 1, wherein the Wnt signaling pathway-associated disease is cancer.

7. The method of claim 6, wherein the cancer is selected from the group consisting of small-cell lung cancer, non-small-cell lung cancer, breast cancer, prostate cancer, carcinoid, bladder cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, renal cancer, head cancer, bone cancer, neck squamous cell carcinoma, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma, thyroid cancer, desmoids, acute myelocytic leukemia (AML), and chronic myelocytic leukemia (CML).

8. The method of claim 1, wherein the antibody is specific for a protein represented by an amino acid sequence having a homology of 100% to the amino acid sequence of (i) or (ii).

9.-31. (canceled)

32. A method for screening a cancer therapeutic agent, the method comprising determining that a candidate substance for cancer treatment is a co-agent for cancer therapy when expression of WLS protein is inhibited in a case where an antibody specific for a WLS (Wntless) motif protein represented by any one of SEQ ID NOs: 1 to 3 is administered in combination with the candidate substance, compared to a case where the antibody for the WLS motif protein is administered alone.

33.-36. (canceled)

37. The method of claim 1, wherein preventing or treating the Wnt signaling pathway-associated disease comprises preventing overactivation of the Wnt signaling pathway to inhibit angiogenesis.

38. The method of claim 37, wherein inhibiting angiogenesis comprises preventing and/or treating cancer growth or metastasis.

39. The method of claim 38, wherein the cancer is selected from the group consisting of small-cell lung cancer, non-small-cell lung cancer, breast cancer, prostate cancer, carcinoid, bladder cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, renal cancer, head cancer, bone cancer, neck squamous cell carcinoma, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma, thyroid cancer, desmoids, acute myelocytic leukemia (AML), and chronic myelocytic leukemia (CML).

40. The method of claim 1, wherein the antibody is specific for a protein represented by an amino acid sequence having a homology of 100% to the amino acid sequence of (i) or (ii).

41. A method for diagnosing cancer or cancer metastasis, the method comprising measuring an expression level of a protein in a subject by use of an antibody specific for the protein represented by an amino acid sequence selected from the group consisting of: (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3; (ii) an amino acid sequence comprising at least 6 consecutive amino acids of the amino acid sequence of any one of SEQ ID NOs: 1 to 3; and (iii) an amino acid sequence having a homology of at least 90% to the amino acid sequence of (i) or (ii).

42. The method of claim 41, wherein the protein is represented by SEQ ID NO: 2.

43. The method of claim 41, wherein the cancer is selected from the group consisting of small-cell lung cancer, non-small-cell lung cancer, breast cancer, prostate cancer, carcinoid, bladder cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, renal cancer, head cancer, bone cancer, neck squamous cell carcinoma, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma, thyroid cancer, desmoids, acute myelocytic leukemia (AML), and chronic myelocytic leukemia (CML).

Description

DESCRIPTION OF DRAWINGS

[0088] FIG. 1 is a schematic view showing the WLS protein before or after Wnt binding according to an example of the present invention.

[0089] FIG. 2 is a schematic view showing the Wnt binding motif of the WLS protein according to an example of the present invention.

[0090] FIG. 3 shows the results of measuring the correlation between WLS expression and gastric cancer prognosis according to an example of the present invention.

[0091] FIG. 4 shows the WLS protein divided into five peptides according to an example of the present invention.

[0092] FIG. 5a shows the results of measuring the responsiveness of WLS#2, WLS#3, WLS#4 and WLS#5 antibodies to the WLS protein in an AGS gastric cancer cell line according to an example of the present invention.

[0093] FIG. 5b shows the results of examining the reduction in WLS detection ability of an antibody used together with a peptide, according to an example of the present invention.

[0094] FIG. 6a shows the results of examining the effect of siWLS #1 on the inhibition of FLAG-WLS expression according to an example of the present invention.

[0095] FIG. 6b shows the results of examining the binding of a monoclonal antibody to immunoprecipitated FLAG-WLS protein according to an example of the present invention.

[0096] FIG. 7a shows the results of examining the position of WLS in an AGS gastric cancer cell line by immunostaining according to an example of the present invention.

[0097] FIG. 7b shows the results of analyzing the expression patterns of WLS in intracellular fractions by Western-blotting according to an example of the present invention.

[0098] FIG. 7c shows the results of examining the position of WLS in an AGS gastric cancer cell line by immunostaining according to an example of the present invention.

[0099] FIG. 7d shows the results of analyzing the binding of WLS#3 antibody to endogenous WLS in an AGS gastric cancer cell line by FACS analysis according to an example of the present invention.

[0100] FIG. 7e shows the results of analyzing the binding of WLS#4 antibody to endogenous WLS in an AGS gastric cancer cell line by FACS analysis according to an example of the present invention.

[0101] FIG. 8 shows the results of analyzing the expression levels of WLS, Axin2 and CyclinD1 in various gastric cancer cell lines according to an example of the present invention.

[0102] FIG. 9 shows the results of analyzing cell viability when an AGS gastric cancer cell line was treated with 1 μg/ml of a WLS monoclonal antibody, according to an example of the present invention.

[0103] FIG. 10a shows the results of analyzing reduced cell viability when an AGS gastric cancer cell line was treated with 4 μg/ml of a WLS monoclonal antibody, according to an example of the present invention.

[0104] FIG. 10b shows the results of observing abnormal cell morphology when an AGS gastric cancer cell line was treated with a WLS monoclonal antibody, according to an example of the present invention.

[0105] FIG. 11 shows the results of examining the effects of WLS#3 and WLS#4 antibodies on the inhibition of cancer cell survival according to an example of the present invention.

[0106] FIG. 12 shows the results of examining cell viability when two gastric cancer lines showing different expression patterns of the WLS gene were treated with a monoclonal antibody, according to an example of the present invention.

[0107] FIG. 13a shows the results of measuring the expression levels of WLS in MKN74 and NI3-3 cells by qRt-PCR according to an example of the present invention.

[0108] FIG. 13b shows the results of measuring the expression levels of WLS in MKN74 and NI3-3 by Western blotting according to an example of the present invention.

[0109] FIG. 13c shows the results of measuring the expression levels of Wnt target genes in MKN74 and NI3-3 cells according to an example of the present invention.

[0110] FIG. 14 shows the results of examining cell viability when MKN74 and NI3-3 cells were treated with a WLS monoclonal antibody, according to an example of the present invention.

[0111] FIG. 15 shows the results of examining the change in cell mobility when expression of WLS was inhibited, according to an example of the present invention.

[0112] FIG. 16 shows the results of examining the effect of a WLS#4 monoclonal antibody on the inhibition of cancer cell mobility according to an example of the present invention.

[0113] FIG. 17 shows the results of examining the effect of a WLS#3 monoclonal antibody on the inhibition of cancer cell mobility according to an example of the present invention.

[0114] FIG. 18 shows the results of FACS analysis that separated MKN74 cells based on the expression level of WLS, according to an example of the present invention.

[0115] FIG. 19 shows the results of FACS analysis that separated AGS cells based on the expression level of CD44, according to an example of the present invention.

[0116] FIG. 20 shows the results of analyzing the expression level of WLS in AGS cells having high CD44 expression, according to an example of the present invention.

[0117] FIG. 21 shows the results of analyzing the acetylation degree of β-catenin in colorectal cancer cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

[0118] FIG. 22a shows the results of measuring the tumor size of MKN74 cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

[0119] FIG. 22b shows the results of measuring the tumor size of MKN74 cells after treatment with a WLS#3 monoclonal antibody according to an example of the present invention.

[0120] FIG. 23 shows the results of examining the mRNA expression levels of WLS in p-MKN74 cells and s-MKN74 with metabolic starvation according to an example of the present invention.

[0121] FIG. 24 shows the results of measuring the tumor sizes of p-MKN74 cells and s-MKN74 with metabolic starvation according to an example of the present invention.

[0122] FIG. 25a shows the results of measuring the tumor sizes of p-MKN74 cells and s-MKN74 after treatment with WLS#3 and WLS#4 monoclonal antibodies according to an example of the present invention.

[0123] FIG. 25b shows the results of measuring the tumor size of p-MKN74 cells after treatment with WLS#3 and WLS#4 monoclonal antibodies according to an example of the present invention.

[0124] FIG. 25c shows the results of measuring the tumor size of s-MKN74 after treatment with WLS#3 and WLS#4 monoclonal antibodies according to an example of the present invention.

[0125] FIG. 26 shows the results of examining the expression level of β-catenin in MCF7 cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

[0126] FIG. 27 shows the results of examining the expression level of β-catenin in WiDr cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

[0127] FIG. 28 shows the results of examining the expression level of β-catenin in A431 cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

[0128] FIG. 29 shows the results of examining the expression level of β-catenin in transfected MCF7 cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

[0129] FIG. 30 shows the results of examining the expression level of β-catenin in transfected WiDr cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

[0130] FIG. 31 shows the results of examining the expression level of β-catenin in transfected A431 cells after treatment with a WLS#4 monoclonal antibody according to an example of the present invention.

BEST MODE

[0131] The present invention provides a pharmaceutical composition for preventing or treating a Wnt signaling pathway-associated disease, the composition containing an antibody specific for a protein represented by an amino acid sequence selected from the group consisting of: (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3; (ii) an amino acid sequence comprising at least 6 consecutive amino acids of the amino acid sequence of any one of SEQ ID NOs: 1 to 3; and (iii) an amino acid sequence having a homology of at least 90% to the amino acid sequence of (i) or (ii).

MODE FOR INVENTION

[0132] Hereinafter, the present invention will be described in further detail. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Identification of WLS Gene

[0133] In order to screen target genes in the early stage of gastric cancer, the normal tissue and cancer tissue of 78 early gastric cancer patients who underwent gastrectomy were comparatively analyzed by a microarray. The results of the analysis indicated that a difference in prognosis appeared in the group in which the expression level of a specific gene was high or low. In particular, in the case of the WLS gene, the recurrence of cancer more occurred in the group in which the WLS gene was highly expressed, and the prognosis was bad in the group. In order to confirm the statistical significance of such results, 506 gastric cancer patients independent of the above group were divided into a group in which the expression of WLS was high and a group in which the expression of WLS was low, and the prognosis of the patients was observed. As a result, it was shown that the group in which WLS was highly expressed had a higher hazard ratio, and this difference in hazard ratio was determined to be statistically significant, based on Cox analysis performed according to age, sex and the degree of cancer progression (stage of cancer progression) (FIG. 3).

Example 2: Construction of WLS Monoclonal Antibodies

[0134] Among portions of the WLS protein structure, which can act as antigens (targeting a portion known as the Wnt-binding region), a total of five peptides (peptide Nos. 1, 2, 3, 4 and 5) were selected. The selected peptides are shown in FIG. 4 and Table 1 below.

TABLE-US-00001 TABLE 1 Amino acid Peptide Nos. positions Amino acid sequences Peptide No. 1 118-137 IAFKLNNQIRENAEVSMDVS Peptide No. 2 138-157 LAYRDDAFAEWTEMAHERVP Peptide No. 3 146-165 AEWTEMAHERVPRKLKCTFT (SEQ ID NO. 2) Peptide No. 4 163-181 TFTSPKTPEHEGRYYECDV (SEQ ID NO. 3) Peptide No. 5 202-222 PVNEKKKINVGIGEIKDIRL

[0135] Construction of antibodies for the five peptides was attempted. Because peptide No. 1 was not produced, production of an antibody for peptide No. 1 was impossible. Using peptide Nos. 2 to 5, a total of four monoclonal antibodies were constructed. The name of each of the antibodies is shown in Table 2 below.

TABLE-US-00002 TABLE 2 Antibody names (monoclonal antibodies) Peptide Nos. WLS#2 Peptide No. 2 WLS#3 Peptide No. 3 (SEQ ID NO: 2) WLS#4 Peptide No. 4 (SEQ ID NO: 3) WLS#5 Peptide No. 5

Example 3: Verification of WLS Monoclonal Antibodies

[0136] The whole sequence of human WLS (NM_024911) was cloned into a pSGS-KF2M1 (FLAG tag in front) plasmid vector and a pSG-KM1F2 (FLAG tag at back) plasmid vector, and a pSGS-empty vector was used as a negative control. FLAG-WLS was expressed in an AGS gastric cancer cell line, and then analyzed by Western blotting. 15 μg of the cell lysate sample was electrophoresed on SDS-PAGE gel, and WLS#2 to WLS#5 monoclonal antibodies were diluted at a ratio of 1:500 vol % with 5 wt % skim milk before use. It was shown that WLS#2 to WLS#5 antibodies detected the FLAG-tagged WLS protein (FIG. 5a). It was observed that this specific reaction was not detected on Western blotting when the WLS#4 antibody was used for treatment together with the peptide (FIG. 5b).

Example 4: Analysis of Endogenous WLS Protein

[0137] Five siRNAs targeting the human WLS gene were constructed (using Genolution pre-designed siRNA service). The results of Western blotting analysis using the WLS#4 antibody indicated that siWLS #1 (sense 5′ GUCAUCUUCUUCAUCGUUAUU 3′; antisense 5′ UAACGAUGAAGAAGAUGACUU 3′) of these siRNAs reduced the expression of FLAG-WLS (FIG. 6a). Furthermore, the FLAG-WLS protein was immunoprecipitated using FLAG-M2 beads, and then specific binding was examined using the WLS monoclonal antibody (WLS#4) (FIG. 6b).

[0138] WLS was cloned into a pEGFP-N1 plasmid vector and expressed in an AGS gastric cancer cell line, and the position thereof in the cells was examined. The results of immunofluorescence staining of the cells using the WLS#4 antibody indicated that WLS was widely distributed in the cytosol (FIG. 7a). Furthermore, untagged WLS protein was expressed with FLAG-WLS and compared, and as a result, the position of endogenous WLS on Western blotting could be predicted. Using the siRNA constructed and analyzed as described above, WLS was lowly expressed in an AGS gastric cancer cell line, and then the intracellular fractions were divided into the cytosol and the cell membrane, and the expression pattern of WLS was analyzed by Western blotting. As a result, it was shown that endogenous WLS was expressed in the cell membrane fraction (FIG. 7b).

[0139] Similarly to the expression patterns of the pEGFP-WLS protein as described above, the AGS gastric cancer cell line was examined by fluorescence staining using the WLS#3 and WLS#4 antibodies, and as a result, it was shown that the endogenous WLS protein was present in the cell membrane (FIG. 7c). Furthermore, the cells were analyzed by FACS analysis, and as a result, it was shown that the WLS#3 and WLS#4 antibodies did bind specifically to the endogenous WLS in the AGS gastric cancer cell line (FIGS. 7d and 7e).

Example 5: Analysis of Gastric Cancer Cell Line

[0140] In order to apply the above-constructed monoclonal antibodies to cell experiments, gastric cancer cells were selected, and RNA was extracted from the selected AGS, SNU-638, SNU-668, KATOIII, MKN28, MKN45 and MKN74 cell lines. cDNA was synthesized from the extracted RNA. Then, using a qRT-PCR technique, the mRNA expression levels of Axin2 and CyclinD1 (which are WLS- and Wnt-targeting genes) in the cells were analyzed. As a result, it was shown that the expression levels of WLS, Axin2 and CyclinD1 in the AGS cell line were commonly high, and that these expression levels were commonly low in the SNU-668, KATOIII, MKN28, MKN45 and MKN74 cells. Exceptionally, in the SNU-638 cells, it was shown that the expression level of WLS was high, but the expression level of the Wnt-targeting gene was low (FIG. 8).

Example 6: Examination of the Ability of WLS Monoclonal Antibodies to Reduce Cell Growth

[0141] The AGS gastric cancer cell line having high expression levels of WLS and Wnt-targeting genes was treated with 0, 0.5 and 1 μg/ml of the WLS monoclonal antibody (WLS#4), and after 48 hours, the viability of the cells was analyzed by an MTT assay (FIG. 9). As a result, when the cells were treated with 1 μg/ml of the antibody, the viability thereof decreased. Furthermore, when the specificity of 1 μg/ml of the antibody was reduced by the peptide, the viability of the cells did not decrease, indicating that the specific binding of the antibody leads to a decrease in the viability of the cells. Additionally, in order to examine the ability of the monoclonal antibody to reduce cell viability, the cells were treated with the antibody while the concentration of the antibody was increased until the cell viability decreased to 50 vol %. As a result, it was shown that, at a WLS#4 antibody concentration of about 4 μg/ml, a decrease in cell viability of about 50 vol % appeared (FIG. 10a). Furthermore, when the AGS gastric cancer cell line was treated with the WLS monoclonal antibody (WLS#4), abnormal morphology appeared, unlike the control group (FIG. 10b). The cells were treated with 2 μg/ml of each of the WLS monoclonal antibodies #3 and #4, and the viability of the cells was analyzed, and as a result, it was shown that the WLS monoclonal antibodies #3 and #4 all significantly reduced the viability of the cancer cells (FIG. 11).

Example 7: Examination of Specific Binding of WLS Monoclonal Antibody and the Ability to Reduce Cell Viability

[0142] Using AGS gastric cancer cells having a high expression level of the WLS genes and MKN45 cells having a low expression level of the WLS gene, the cells were treated with the WLS#4 monoclonal antibody, and the viability of the cells was analyzed. As a result, the monoclonal antibody was effective even when WLS was highly expressed (FIG. 12). Because this phenomenon is attributable to the features of the AGS and MKN45 cells themselves, a cell model having the same genetic background was prepared. Specifically, MKN74 cells having a low expression level of the WLS gene were injected into the heart of a rat, and the cells were allowed to metastasize to the brain of the rat, after which the MKN74 cells grown in the brain were cultured and named “NI3-3”. The expression levels of the WLS gene in the two cell types were analyzed by qRt-PCR and Western blotting using the WLS#4 antibody, and as a result, it was shown that the expression level of WLS in the cells that metastasized to the brain cells was higher (FIGS. 13a and 13b). Furthermore, it was shown that the activities of WLS, Axin2 and CyclinD1 genes (which are intracellular Wnt-targeting genes) in the metastasized cells increased compared to those in the original MKN74 cells (FIG. 13c). The ability of the WLS#4 monoclonal antibody to reduce cell viability was examined in the same manner as described above, and as a result, it was shown that the cell viability of only the NI3-3 cell line having a high expression level of WLS was reduced, and it was shown by a peptide inhibitory experiment that this reduction in cell viability was caused by the specific binding of the antibody (FIG. 14).

Example 8: Examination of the Ability of WLS Monoclonal Antibodies to Reduce Cell Migration

[0143] The Wnt gene was expressed in the AGS gastric cancer cell line, and then the mobility of the cells was examined by a cell migration experiment (scratch and healing). The AGS gastric cancer cells were cultured to confluency in a culture dish, and then the cell monolayer was linearly scratched using a 200-μl tip, after which the area in which the cells migrated for 6 hours was calculated. As a result, it was shown that when the expression of WLS was reduced using siWLS #1, the mobility of the cells was reduced (FIG. 15). In addition, using siRNA as a control, the ability of the WLS#4 monoclonal antibody to reduce cell mobility was measured, and as a result, it was shown that the mobility of the cells was also reduced in the group treated with the WLS#4 monoclonal antibody (FIG. 16), similar to the group in which the expression of WLS was reduced by siRNA. In addition, similarly to the WLS#4 monoclonal antibody, it was shown that the WLS#3 monoclonal antibody also reduced the mobility of the cells when the Wnt gene in the cancer cells was expressed (FIG. 17).

Example 9: FACS Analysis of WLS Monoclonal Antibody

[0144] The results of the above Examples indicated that the high expression of WLS can adversely affect the prognosis of the gastric cancer patients and can lead to the increase in metastasis ability in the cell experiment. Thus, in order to analyze the features of cells in which WLS is highly expressed, FACS analysis was performed. Through FACS analysis using the WLS#4 monoclonal antibody, cells having a high expression level of WLS and cells having a low expression level of WLS were separated from MKN74 cells (FIG. 18). Moreover, using the protein CD44 that determines the tumorigenicity of gastric cancer cells, AGS cells were analyzed by FACS analysis (FIG. 19). As a result, it was shown that the expression level of WLS in the AGS cells having a high level of CD44 was also high (FIG. 20).

Example 10: Inhibition of Signaling of Colorectal Cancer Caused by Overactivation of Wnt Signaling Pathway

[0145] The gastric cancer cell line AGS and the HT29 and SW480 colorectal cancer lines caused by overactivation of the Wnt signaling pathway were treated with the WLS#4 monoclonal antibody constructed in Example 2, and then a change in the signaling was observed. Specifically, each of the cell lines was treated with 0, 1, 2 and 4 μg/ml of the WLS#4 monoclonal antibody for 48 hours, and then the acetylation of the major transcriptional regulator β-catenin of the Wnt signaling pathway was analyzed by Western blotting. As a result, it was shown that the acetylation of (3-catenin that increased due to overactivation of the Wnt signaling pathway was reduced by treatment with the WLS#4 monoclonal antibody (FIG. 21).

Example 11: Examination (1) of Anticancer Effect of WLS Monoclonal Antibodies

[0146] A total of 1×10.sup.4 MKN45 cells mixed with Matrigel at a ratio of 1:2 (v/v) were injected into the subcutaneous tissue of immunodeficient nude mice to form a gastric tumor. When the volume of the tumor reached 100 mm.sup.3 after about 2 weeks, the WLS monoclonal antibodies constructed in Example 2 were injected through the tail veins. The WLS#4 monoclonal antibody was injected at a concentration of 5 mg/kg six times for 2 weeks (n=8), and the WLS#3 monoclonal antibody was injected at a concentration of 5 mg/kg nine times for 3 weeks (n=20). During the period from the day on which the monoclonal antibody was first injected to two days following the day on which the monoclonal antibody was finally injected, the volume of the tumor was measured using calipers according to the following equation: short axis.sup.2×long axis/2. As a result, it was shown that the tumor size in the test group treated with the WLS#3 or WLS#4 monoclonal antibody significantly decreased compared to the control group not treated with the antibody (FIGS. 22a and 22b).

Example 12: Examination (2) of Anticancer Effect of WLS Monoclonal

[0147] Antibodies

[0148] The MKN45 cell line was metabolically starved to establish a selected MKN45 cell line (s-MKN45). The mRNA level of WLS in such s-MKN45 cells was analyzed by a qRT-PCR experiment, and as a result, it was shown that the expression level of WLS in the s-MKN45 cells was higher than that in a non-starved control group (p-MKN45) (FIG. 23). A total of 1×10.sup.7 p-MKN45 cells or s-MKN45 cells mixed with Matrigel at a ratio of 1:2 (v/v) were injected into the subcutaneous tissue of immunodeficient nude mice to form a gastric tumor, and as a result, it was shown that the tumorigenicity of the s-MKN45 cell line was higher than that of the p-MKN45 cell line (FIG. 24). In addition, a total of 4×10.sup.6 p-MKN45 cells or s-MKN45 cells mixed with Matrigel at a ratio of 1:2 (v/v) were injected into the subcutaneous tissue of immunodeficient nude mice to form a gastric tumor, and when the volume of the tumor reached 100 mm.sup.3 after about 2 weeks, the monoclonal antibodies constructed in Example 2 were injected through the tail veins. 100 μl of each of the WLS#3 and WLS#4 monoclonal antibodies was injected at a concentration of 1 mg/ml three times for 2 weeks. During the period from the day on which the monoclonal antibody was first injected to two days following the day on which the monoclonal antibody was finally injected, the volume of the volume was measured using calipers according to the following equation: short axis.sup.2×long axis/2. As a result, it was shown that the tumor size in both the p-MKN45 cell line and the s-MKN45 cell line in the test group treated with the WLS#3 or WLS#4 monoclonal antibody significantly decreased compared to that in the control group not treated with the antibody (FIGS. 25a to 25c).

Example 13: Examination of the Change in P-Catenin Expression Caused by WLS Monoclonal Antibody

[0149] The breast cancer cell line MCF7, the colorectal cancer cell line WiDr and the skin cancer cell line A431 were treated with the WLS#4 monoclonal antibody constructed in Example 2, and after 72 hours, a change in the expression level of β-catenin in each cell line was analyzed by real-time PCR. As a result, it was shown that the expression of β-catenin in all the breast cancer cell line, the colorectal cancer cell line and the skin cancer cell line was reduced by treatment with the WLS#4 monoclonal antibody (FIGS. 26 to 28).

Example 14: Examination of the Change in P-Catenin Expression Caused by WLS Monoclonal Antibody

[0150] The breast cancer cell line MCF7, the colorectal cancer cell line WiDr and the kidney cell line 293T were transfected with a Wnt3a-pcDNA3.1 plasmid, and after 24 hours, each of the transfected cell lines was treated for 48 hours with the WLS#4 monoclonal antibody constructed in Example 2. After treatment, a change in the expression level of β-catenin in each cell line was analyzed by real-time PCR.

[0151] The expression of β-catenin in the breast cancer cell line is as reported in various publications (Carcinogenesis. 2000 July; 21(7):1453-6). Although the expression level of β-catenin in the MCF7 cells was also observed to be high, it was shown that the expression level of β-catenin in the MCF7 cells was reduced by treatment with the WLS#4 monoclonal antibody (FIG. 29).

[0152] The expression of β-catenin in the colorectal cancer cell line is as reported in various publications (Nature Communications 4, Article number: 2610). Although it could be seen that the expression level of β-catenin in the WiDr cells was also observed to be high and the expression level further increased by continuous stimulation of the Wnt protein, it was shown that the expression level of β-catenin in the WiDr cells was reduced by treatment with the WLS#4 monoclonal antibody (FIG. 30).

[0153] It was reported that abnormalities in the Wnt signaling pathway in kidney tissue cause various diseases, including renal cancer (Organogenesis. 2008 April-June; 4(2): 55-59). In order to confirm this report, it could be seen that the expression level of β-catenin in the 293T cells was also observed to be high and the expression level further increased by continuous stimulation of the Wnt protein, but it was shown that the expression level of β-catenin in the 293T cells was reduced by treatment with the WLS#4 monoclonal antibody (FIG. 31).

Example 15: Epitope Mapping for WLS Monoclonal Antibody

[0154] Epitope mapping of the peptide (peptide No. 4) for the WLS#4 monoclonal antibody constructed in Example 2 was performed. The amino acid sequence of peptide No. 4 was divided into three amino acid fragments (recombinant peptide Nos. 4-1 to 4-3) as shown in Table 3 below, and each of the fragments was bound to a SNCA-6×His target to thereby construct recombinant peptides. Using Western blotting, each of the recombinant peptides was reacted with the WLS#4 monoclonal antibody, and the peptide bound to the monoclonal antibody was selected. As a result, it was shown that recombinant peptide No. 4-3 did bind to the WLS#4 monoclonal antibody.

TABLE-US-00003 TABLE 3 Amino acid sequences Mapping results for peptide No. 4 TFTSPK Recombinant peptide No. 4-1 TPEHEG Recombinant peptide No. 4-2 RYYECDV Recombinant peptide No. 4-3

INDUSTRIAL APPLICABILITY

[0155] As described above, the present invention is directed to an antibody for preventing or treating a disease associated with the Wnt signaling pathway and to a pharmaceutical composition containing the same.

TABLE-US-00004 [Sequence List Text] SEQ ID NO. 1:  AEWTEMAHERVPRKLKCTFTSPKTPEHEGRYYECDV SEQ ID NO. 2:  AEWTEMAHERVPRKLKCTFT SEQ ID NO. 3:  TFTSPKTPEHEGRYYECDV SEQ ID NO. 4:  TFTSPK SEQ ID NO. 5:  TPEHEG SEQ ID NO. 6:  RYYECDV