WO2009120039A2

WO2009120039A2 - Use of cd9 for a target protein to develop anticancer drug of solid cancers that overexpress cd9

Info

Publication number: WO2009120039A2
Application number: PCT/KR2009/001576
Authority: WO
Inventors: Je Ho Lee; Jae Ryoung Hwang
Original assignee: Sungkyunkwan University Foundation For Corporate Collaboration
Priority date: 2008-03-27
Filing date: 2009-03-27
Publication date: 2009-10-01
Also published as: KR20090103163A; KR101008314B1; WO2009120039A3

Abstract

Disclosed is the use of CD9 for a target protein to develop an anticancer agent of solid cancer that overexpresses CD9. Since CD9 is overexpressed in solid cancer cells and functions to augment cell viability, invasion and migration required for the growth of solid cancer cells, it may be useful as a target protein for developing an anticancer agent of solid cancer.

Description

[DESCRIPTION] [Invention Title]

USE OF CD9 FOR A TARGET PROTEIN TO DEVELOP ANTICANCER DRUG OF SOLID CANCERS THAT OVEREXPRESS CD9

[Technical Field]

This disclosure relates to the use of CD9 for a target protein for developing an anticancer agent of solid cancer that overexpresses CD9. CD9 is useful as a target protein for developing an anticancer agent of solid cancer due to its overexpression in solid cancer cells and its diverse functions required for the growth of the solid cancer cells.

[Background Art]

CD9 is a cell membrane glycoprotein receptor that belongs to the tetraspanin family, and has a molecular weight of about 24-27 kDa. In addition, it is known that CD9 is involved in a wide range of phenomena occurring in cells, for example, cell proliferation (Higashiyama, S., et a/., J. Cell. Biol. 128:929-938, 1995; Iwamoto, R., et a/., EMBO J. 13:2322-2330 1994), adhesion (Anton, E. S., et al., J. Neurosci. 15:584-595 , 1995; Hadjiargyrou, M., et a/., J. Neurosci. 15:574-583 , 1995; Masellis Smith, A., and Shaw, A. R., J. Immunol. 152:2768- 2777, 1994), metastasis (Ikeyama, S., et a/., J. Exp. Med. 177:1231-1237, 1993), and migration (Klein-Soyer, C, et al., Arterioscler Thromb Vase Biol. 20:360-369, 2000). However, studies about the molecular mechanisms of such CD9 functions are not sufficiently conducted yet.

Testraspanins have four transmembrane domains and two extracellular loops. The transmembrane domains are substantially conserved among the proteins that belong to the same family. Tetraspanins are also called 'tetraspanin web', since they are known to interact with integrins, membrane- anchored growth factors and the same tetraspanin family (Radford, K. J., et al., Biochem. Biophys. Res. Comm. 222:13-18, 1996; Iwamoto, R., et al., EMBO J. 13:2322-2330 1994; Rubinstein, E., et al., Eur. J. Immunol. 24:3005-3013, 1994; Le Naour, F., et al., MoI. Cell. Proteomics 5:845-857, 2006). Therefore, it is expected that tetraspanins may function as cell surface molecule organizers or as facilitators capable of facilitating the formation or stability of functional signaling complexes (Wright, M. D. and Tomlinson, M. G. Immunol Today 15:588-594, 1994). CD9 is expressed in most cells. CD9 genes were cloned and their structure was identified in 1991 (Miyake, M. et ai, J. Exp. Med. 174: 1347-1354, 1991). Then, in vivo CD9 functions were studied through CD9 knockout mice in 2000 (Kaji, K., et a/., Nat Genet 24:327-334, 2000; Le Naour, F., et a/., Science 287:319-321 , 2000). Particularly, it has been known that CD9 exists in ovum cell membrane to facilitate fertilization between ovum and sperm. It has also been known that CD9 is expressed in pre-B cells and blood platelets and is involved in the platelet-induced endothelial cell proliferation (Masellis Smith, A., and Shaw, A. R., J. Immunol. 152:2768-2777, 1994).

According to the studies of CD9 in cancer, CD9, also called motility- related protein (MRP)-I , has been reported to be involved in cell motility and tumor metastasis (Miyake, M. and Hakomori, S., Biochemistry 30:3328-3334, 1991). It has been reported that CD9 antibodies inhibit the cell motility in several cancer cell lines, and transfection of CD9 cDNA into cancer cells suppresses cell motility and growth (Ikeyama, S., et a/., J. Exp. Med. 177:1231- 1237, 1993). It has been also reported that the degree of CD9 expression is in inverse proportion to the degree of cancer progress. For example, as known in the art, CD 9 expression is decreased in patients suffering from colon cancer (Mori, M., et al., CHn. Cancer Res. 4:1507-1510, 1998), breast cancer (Miyake, M., et a/., Cancer Res. 55:4127-4131 , 1995), lung cancer (Higachiyama, M., et al., Cancer Res. 55:6040-6044, 1995; Funakoshi, T., et a/., Oncogene 22:674- 687, 2003) and pancreatic cancer (Sho, M., et a/., /nf. J. Cancer 79:509-516, 1998), and such decreased CD9 expression is related to invasion, metastasis and bad prognosis. On the other hand, it has been reported that CD9 expression increases in proportion to the degree of cancer progress, in the cases of head and neck squamous cell carcinoma (Erovic, B. M., et al., Head Neck 25:848-857, 2003) and gastric cancer (Hori, H., et al., J. Surg. Res. 117:208-215, 2004). It is estimated from such contradictory reports about CD9 that CD9 is tissue-specific. Meanwhile, CD9 expression in ovarian cancer has been studied through techniques based on microarray or immunohistochemistry (Drapkin, R., et al., Hum Pathol. 35:1014-1021 , 2004; Peters, D. G., et al., Cancer Epidemiol Biomarkers Prev. 14:1717-1723, 2005; Houle, C. D., et al., Gynecol Oncol 86:69-78, 2002). However, the results of such studies conflict with each other. Moreover, there is no study about the functions of CD9 in ovarian cancer. Under such circumstances, after CD9 expression and CD9 functions are analyzed according to this disclosure, it is shown that CD9 is overexpressed in ovarian cancer, functions to enhance cell viability, invasion and migration capability required for the growth of the solid cancer cells, and serves to activate NF-κB signaling system. Thus, CD9 may be used as a target protein for developing an anticancer agent of solid cancer.

[Disclosure]

[Technical Problem]

Disclosed is an anticancer agent including a CD9 inhibitor as an active ingredient. Disclosed also is a method for treating cancer by using the CD9 inhibitor.

[Technical Solution]

In one aspect, there is provided an anticancer agent including a CD9 inhibitor as an active ingredient. In another aspect, there is provided a method for treating cancer, which includes administrating a pharmaceutically effective amount of the anticancer agent to a subject suffering from cancer.

In still another aspect, there is provided a method for tracing cancer, which includes administering an antibody that binds specifically to CD9 to a subject, and imaging the antibody.

In still another aspect, there is provided a kit for diagnosing cancer, which includes an antibody that binds specifically to CD9.

In yet another aspect, there is provided a method for diagnosing cancer, which includes measuring the amount of CD9 expression in cancer candidate tissue-derived cells; and comparing the amount of CD9 expression with the amount of CD9 expression in normal tissue-derived cells to select tissues in which CD9 expression is increased.

[Advantageous Effects]

CD9 is overexpressed in solid cancer cells, and functions to enhance cell viability, invasion and migration capability required for the growth of the solid cancer cells. Thus, CD9 may be used as a target protein for developing an anticancer agent of solid cancer. In addition, a CD9 inhibitor effectively inhibits the functions of CD9 in cancer cells to inhibit growth, invasion and migration of cancer cells, and suppresses NF-κB signaling, so that it may be useful for anticancer treatment.

[Description of Drawings]

The above and other aspects, features and advantages of the disclosed exemplary embodiments will be more apparent from the following detailed description taken in conjunction with the accompanying drawings in which:

Fig. 1 illustrates CD9 gene expression in ovarian cancer tissues as determined by microarray analysis;

Fig. 2 is a graph showing CD9 gene expression in ovarian cancer tissues as determined by microarray analysis, wherein P represents patients suffering from ovarian cancer, N represents CD9 expression in normal tissues and T represents CD9 expression in cancer tissues;

Fig. 3 is a graph showing CD9 gene expression in ovarian cancer tissues as determined by real-time polymerase chain reaction (PCR), wherein N represents normal tissues, B represents borderline tumor, S represents serous type ovarian cancer, and M represents mucinous type ovarian cancer;

Fig. 4 is a graph showing CD9 gene expression in ovarian cancer tissues as determined by microarray analysis, wherein B represents borderline tumor, S represents serous type ovarian cancer, and M represents mucinous type ovarian cancer;

Fig. 5 illustrates CD9 gene expression in ovarian cancer tissues as determined by reverse transcription (RT)-PCR;

Fig. 6 illustrates CD9 protein expression in ovarian cancer tissues as determined by immunohistochemical analysis (600X);

Fig. 7 illustrates CD9 protein expression in ovarian cancer tissues as determined by western blot analysis;

Fig. 8 illustrates CD9 protein expression in ovarian cancer, cervical cancer and breast cancer cell lines as determined by western blot analysis;

Fig. 9 illustrates the location of cell membrane expression of CD9 proteins in ovarian cancer cell line 2774 as determined by immunofluorescence microscopy; Fig. 10 illustrates CD9 protein expression in CD9/SKOV3 stable cell line as determined by western blot analysis;

Fig. 11 is a graph showing the function of CD9 of stimulating cell growth in CD9/SKOV3 cell line as determined by cell viability assay;

Fig. 12 is a graph showing the function of CD9 of stimulating cell growth in 2774 ovarian cancer cell line as determined by cell viability assay using CD9 monoclonal antibody that inhibits CD9 function;

Fig. 13 is a graph showing the function of CD9 of stimulating invasion in 2774 ovarian cancer cell line as determined by invasion assay using CD9 antibody that inhibits CD9 function;

Fig. 14 is a graph showing the function of CD9 of stimulating migration in 2774 ovarian cancer cell line as determined by migration assay using CD9 antibody that inhibits CD9 function;

Fig. 15 shows the results of western blot analysis in CD9/SKOV3 cell line, wherein p65, a subunit of NF-κB, migrates highly toward the nucleus as compared to Vec/SKOV3 (Cyt: cytoplasmic fraction; Nuc: nuclear fraction);

Fig. 16 shows the results of immunofluorescence microscopy in CD9/SKOV3 cell line, wherein p65, a subunit of NF-κB, migrates toward the nucleus, which demonstrate activation of NF-κB signaling (arrow head (Δ) represents p65 in cytoplasm, and arrows (→) represent p65 in the nucleus);

Fig. 17 is a photographic view showing the inhibition of cancer growth in the mice to which ALB6, a CD9 antibody that inhibits CD9 functions, is injected, as compared to the mice to which PBS or IgGI is injected;

Fig. 18 is a bar graph showing the results of measuring the tumor weights obtained from the experiment as shown in Fig. 17; and

Fig. 19 shows the results of immunohistochemical staining assay of tumors obtained from each mouse using mouse secondary antibodies alone, which demonstrate that ALB 6 is well transferred to the tumors.

[Best Mode]

Exemplary embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments are shown. This disclosure may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth therein. Rather, these exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of this disclosure to those skilled in the art. In the description, details of well- known features and techniques may be omitted to avoid unnecessarily obscuring the presented embodiments.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the use of the terms a, an, etc. does not denote a limitation of quantity, but rather denotes the presence of at least one of the referenced item. It will be further understood that the terms "comprises" and/or "comprising", or "includes" and/or "including" when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

In one aspect, there is provided the use of CD9 as a target protein for diagnosing and tracing cancer.

Herein, the cancer may include breast cancer, cervical cancer or ovarian cancer, more specifically ovarian cancer, but not limited thereto. The cancer may include all types of cancer in which CD9 is overexpressed.

In this context, microarray analysis of gene expression in normal tissues and ovarian cancer tissues is carried out to find genes capable of targeting ovarian cancer. As a result, in overall ovarian cancer tissues regardless of the particular type of ovarian cancer, CD9 gene expression increases to 4-10 times of gene expression in normal tissues (see Figs. 1 and 2). Since such specific expression of a large amount of target material in cancer tissues is favorable for specific targeting in cancer cells, it is believed that CD9 may be used as a target material.

To investigate CD9 gene expression in ovarian cancer tissues, normal tissues and ovarian cancer tissues are subjected to real-time PCR and RT-PCR. As a result, CD9 gene expression increases in ovarian cancer tissues as compared to normal tissues. Particularly, CD9 gene expression increases to 5-12 times in borderline tumor and serous type ovarian cancer. Therefore, it is believed that CD9 genes are capable of targeting ovarian cancer (see Figs. 3-5). In addition, to investigate CD9 protein expression in ovarian cancer tissues, immunohistochemical analysis and western blot analysis are carried out in normal tissues and ovarian cancer tissues. As a result, it is shown that CD9 protein is overexpressed in borderline tumor, serous type and mucinous type ovarian cancer and CD9 is expressed in cell membrane of ovarian cancer. These results conform to the location of CD9 in normal cells known to date in the related art (see Fig. 6). In the case of western blot analysis, CD9 protein is overexpressed in serous type ovarian cancer as compared to normal tissues or mucinous type ovarian cancer. In the case of mucinous type ovarian cancer, the result does not conform to the result of CD9 gene expression. Such different results are caused by the sensitivity of each test method. In other words, the test method used for determining gene expression has a higher sensitivity than the western blot analysis. Also, the stability and turnover time of the protein itself may affect the protein expression (see Fig. 7). Additionally, a normal cell line, ovarian cancer cell line, cervical cancer cell line and breast cancer cell line are subjected to western blot analysis to investigate CD9 protein expression in various cultured cancer cell lines. After the analysis, it is shown that CD9 protein expression increases in all types of cancer cell lines as compared to the normal cell line (see Fig. 8).

Further, immunofluoroescence microscopy is carried out to determine the location of CD9 expression in cancer cells. As a result, it is shown that CD9 exists in the cell membrane of a cancer cell line. This suggests that the location of CD9 expression is the same in normal cells and cancer cells. Since a target material desirably exists on the cancer surface to accomplish specific targeting of cancer cells, it is believed that CD9 is useful as a target material (see Fig. 9).

In another aspect, there is provided a method for tracing cancer, which includes administering an antibody that binds specifically to CD9 to a subject, and imaging the location of the antibody.

In the method, the imaging may be performed by using a secondary antibody, or a conjugate, labeled with a chromophoric enzyme, fluorescent material, radioactive isotope or colloid, to perform tracking of the antibody-CD9 conjugate by way of fluorescence, luminescence, chemiluminescence, absorbance, reflection or transmission, but is not limited thereto. The chromophoric enzyme may include peroxidase, alkaline phosphatase or acid phosphatase (e.g. horseradish peroxidase). The fluorescent material may include fluorescein carboxylate (FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetoxycumarin-3-yl, fluorescein-5-yl, fluorescein- 6-yl, 2',7'-dichlorofluorescein-5-yl, 2',7'-dichlorofluorescein-6-yl, dihydrotetramethylrhodamin-4-yl, tetramethylrhodamin-5-yl, tetramethylrhodamin-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a,diaza-s- indacen-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacen-3- ethyl. The radioactive isotope for use in labeling may include but is not limited to Sc-47, Cu-64, Cu-67, Ga-68, Br-76, Y-86, Y-90, Tc-99m, ln-111 , Sm-153, Dy-165, Ho-166, Er-169, Yb-169, Lu-177, Re-186 and Re-188.

In still another aspect, there is provided an anticancer agent including a CD9 inhibitor as an active ingredient.

The anticancer agent disclosed herein inhibits growth, invasion and migration of cancer cells.

To investigate the functions of CD9, cell viability, invasion and migration of cancer cells are determined by applying a CD9 monoclonal antibody, whose antigen is a functional region of CD9, in an ovarian cancer cell line. As a result, it is shown that when the functional region of CD9 is blocked by the antibody, the cell viability, invasion and migration are inhibited (see Figs. 12-14). Therefore, it can be seen that CD9 plays an important role in augmenting such functions required for cancer growth in the ovarian cancer cell line.

In addition, genes up-regulated by CD9 overexpression are observed via microarray analysis to investigate the effect of CD9 on signaling. As a result, it is shown that CD9 overexpression significantly controls expression of various genes related to NF-κB signaling pathway (see Table 1). The NF-κB signaling pathway is one by which NF-κB is activated, and plays an important role in tumorigenesis of cells including cell growth.

Further, a CD9 monoclonal antibody is injected to an ovarian cancer animal model and then the tumor size and survival rate are determined to study the effect of inhibition of CD9 functions on cancer cells. The ovarian cancer animal model is obtained by injecting ovarian cancer 2774 cell line that expresses green fluorescence protein stably to 7 week-aged female nude mice (BALB/C nu/nu) (5X10⁶ cells per mouse) via an intraperitoneal route (Furuya, M., et a/., Cancer Res. 65:2617-2625, 2005; Zvieriev, V., et a/., Biochem. Biophys. Res. Commun. 337:498-504, 2005). One week after the injection of 2774 cells, ALB6, a CD9 antibody, is injected to the abdominal cavity at a dose of 2 mg per kg of mouse body weight with 3-7 day intervals. A mouse antibody (IgGI) is injected as a control. After the antibody is injected four to five times, the mice are sacrificed and the cancer generated in the abdominal cavities is observed. As a result, it can be seen that cancer cell growth is suppressed by inhibiting the functions of CD9. Therefore, it is believed that CD9 overexpressed on the cancer cell surface may be used as an effective target material for solid cancer that overexpresses CD9, including ovarian cancer, to develop an effective anticancer agent.

The CD9 inhibitor may be selected from antisense nucleotides that bind complementarily to mRNA of CD9, small interfering RNA (siRNA) molecules, substrate analogues and antibodies that bind to CD9 protein, and small compounds inhibiting the activities of CD9.

Antisense nucleotides

Antisense nucleotides are currently recognized as therapeutic agents that are expected to be used in treatment of various human diseases. As defined by Watson -C rick base pairing, antisense nucleotides are bound (hybridized) with complementary base sequences of DNA, premature-mRNA or mature-mRNA to inhibit genetic information flow from DNA to protein. Specificity to target sequences of antisense nucleotides makes them exceptionally multifunctional. Because antisense nucleotides are long chains consisting of monomer units, they may be easily synthesized for their target RNA sequences. Many recent studies demonstrate the utility of antisense nucleotides as biochemical agents for the study of target proteins (Rothenberg et ai, J. Natl. Cancer Inst, 81 :1539-1544, 1999). Recently, there is great advancement in the field of oligonucleotide chemistry and synthesis of nucleotides that show improved cell adsorption, targeting affinity and nuclease resistance, and thus the use of antisense nucleotides may be considered as a new type of therapeutic agent. Therefore, antisense nucleotides may be constructed by using mRNA sequences known to those skilled in the art, and it is expected that the antisense nucleotides bind specifically to CD9 mRNA to inhibit CD9 expression. siRNA molecules Provided is a siRNA molecule having a double stranded RNA molecule formed from sense RNA and antisense RNA, wherein the sense RNA includes the same nucleic acid sequence as the target sequence of CD9 mRNA nucleotides. The siRNA molecule may include a sense sequence selected from the base sequence of CD9 and an antisense sequence that binds complementarily to the sense sequence. Any double stranded RNA molecules may be used as long as the RNA molecules have a sense sequence capable of binding complementarily to the base sequence of CD9. More particularly, the antisense sequence may have a sequence complementary to the sense sequence. Inhibitors against the functions of CD9 protein may be selected from the group consisting of peptides, antibodies and substrate analogues that bind to the protein, small compounds inhibiting activities of CD9 protein, etc.

Antibodies

Antibodies against CD9 may bind specifically and directly to CD9 and effectively inhibit activities of CD9. Antibodies that bind specifically to CD9 may include polyclonal antibodies or monoclonal antibodies, more specifically monoclonal antibodies. Antibodies that bind specifically to the biomarker may be obtained by any method known to those skilled in the art. Commercially available antibodies may also be used. The antibodies may be prepared by injecting CD9 protein as an immunogen to an external host according to a known manner. Particular examples of the external host include mammals, such as mice, rats, sheep and rabbits. The immunogen is injected via an intramuscular, intraperitoneal or subcutaneous route. In general, the immunogen may be administered together with an adjuvant for augmenting the antigenicity. The antibodies may be separated by collecting sera that show an improved titer and antigen specificity from blood sampled periodically from the external host.

Substrate analogues

Analogues (such as peptide or non-peptide medicines) inhibiting the binding domain of CD9 may be prepared to inhibit activities of CD9. Particularly, such peptide analogues or non-hydrolyzable peptide analogues of CD9 substrate fragments may be used. The non-hydrolyzable peptide analogues may be produced by using β-turn dipeptide cores (Nagai et ai, Tetrahedron Lett. 26:647, 1985), keto-methylene pseudopeptides (Ewenson et ai, J. Med. Chem. 29:295, 1986; Ewenson et ai, in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium), Pierce Chemical Co. Rockland, IL, 1985), azepines (Huffman et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al., in Peptides; Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), β-aminoalcohols (Gordon et al., Biochem. Biophys. Res. Commun. 126:419 1985) and substituted gamma-lactam rings (Garvey et al., in Peptides: Chemistry and Biology, G R. Marshell ed., ESCOM Publisher: Leiden, Netherlands, 1988).

The anticancer agent disclosed herein includes the CD9 inhibitor as an active ingredient. The anticancer agent may include the CD9 inhibitor in an amount of 0.0001-50 parts by weight based on the total weight thereof. The anticancer agent may further include at least one active ingredient that shows the same or similar activity.

The anticancer agent may be formulated for administration by using at least one pharmaceutically acceptable carrier in addition to the active ingredient. Such pharmaceutically acceptable carriers may include saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome or a combination thereof. If desired, other conventional antioxidants, buffers, bacteriostats, etc. may be added. In addition, diluents, dispersants, binders or lubricants may be further added to provide injection formulations, such as aqueous solution, suspension or emulsion, pills, capsules, granules or tablets. It is also possible to bind such carriers with a target organ-specific antibody or other ligands so that they may act specifically on a target organ. Further, the anticancer agent may be formulated depending on the particular disease to be treated or particular ingredient according to a method known in the related art or a method described in Remington's document (Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, PA).

There is no particular limitation in methods for administering the anticancer agent disclosed herein. The anticancer agent may be administered parenterally (e.g. intravenous, subcutaneous, intraperitoneal or local routes) or orally, as appropriate. Particularly, the anticancer agent may be administered parenterally, more particularly, via intravenous injection. The anticancer agent may be administered in a wide range of doses depending on the patient's body weight, age, sex, physical conditions, diet, administration time, administration method, excrement, disease severity, or the like. The anticancer agent may be administered at a daily dose of about 0.01-12.5 mg/kg, particularly 1.25-2.5 mg/kg, once per day or several times per day at divided dose schedules.

When toxicity test of the anticancer agent was carried out for intravenous injection to mice, LD50, i.e. the amount which causes the death of 50%, was at least 1 ,000 mg/kg. This means that the anticancer agent is safe for the intravenous administration.

The anticancer agent may be used to treat cancer, alone or in combination with surgery, hormone therapy, drug therapy and biological response modifier drug therapy.

In still another aspect, there is provided a method for treating cancer, which includes administering an anticancer agent containing a pharmaceutically effective amount of CD9 inhibitor to a subject suffering from cancer.

The effective amount may be such an amount that the anticancer agent inhibits cancer growth, invasion and migration.

The cancer may include all types of solid cancer that overexpress CD9. Particularly, the cancer may be breast cancer, cervical cancer or ovarian cancer, more particularly ovarian cancer, but is not limited thereto.

The kit disclosed herein may be used in determining CD9 as a protein marker that shows a difference in expression between subjects suffering from cancer and normal subjects. The kit determines whether a subject suffers from cancer or not, thereby permitting medical practitioners to diagnose cancer, and monitors responses of a subject to treatment, thereby permitting a modification in cancer therapy depending on the result.

The kit disclosed herein may be used after treatment with an antibody that binds to the protein marker to monitor of the amount of the antibody bound to the protein marker. In this manner, the kit may be used for diagnosing cancer.

To monitor the amount of the bound antibody, high-throughput screening (HTS) systems may be used. More particularly, as non-limiting examples, fluorescence spectroscopy based on detection of fluorescence caused by a fluorescent marker, surface plasmon resonance (SPR) systems based on real time measurement of a change in plasmon resonance without using any fluorescent label, or surface plasmon resonance imaging (SPRI) systems based on imaging of SPR systems may be used herein. The fluorescence spectroscopy includes labeling an antibody that binds specifically to the protein marker with a fluorescent material, spotting the labeled antibody, and detecting signals by using a fluorescence scanner program. In this manner, the binding degree of the antibody may be observed. The fluorescent material may be selected from the group consisting of Cy3, Cy5, poly-L-lysine, fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC) and rhodamine, but is not limited thereto.

Unlike the fluorescent spectroscopy, the SPR systems allow real time analysis of the binding degree of the antibody with no need of labeling samples with a fluorescent material. In the case of SPRI systems, multiple simultaneous sample analysis may be performed by using microarray analysis.

In addition, the kit disclosed herein may further include a stripping agent or eluent capable of removing a substrate to be subjected to color development with an enzyme and non-bound protein so that only the bound CD9 is left. Samples used for analysis include biosamples that enable determination of disease-specific polypeptides distinct from normal conditions, and particular examples thereof include sera, urine, tears and sputa. More particularly, biological liquid samples, such as blood, sera or plasma may be used for determining the bound CD9. Such samples may be prepared for augmenting the detection sensitivity of the protein marker. For example, serum samples collected from patients may be pretreated by using anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction or gel electrophoresis.

In the method, the amount of CD9 expression in cancer candidate tissue-derived cells may be determined by microarray, real-time PCR and RT- PCR using genes, and immunohistochemical analysis and western blot analysis using proteins. However, methods for determining the amount of CD9 expression are not limited thereto.

The cancer may include but are not limited to breast cancer, cervical cancer or ovarian cancer, more specifically ovarian cancer. The cancer may include all types of cancer in which CD9 is overexpressed. [Mode for Invention]

The examples and experiments will now be described. The following examples and experiments are for illustrative purposes only and not intended to limit the scope of this disclosure.

<Example 1> Examination of CD9 Gene Expression in Ovarian Cancer Tissues

<1-1> Microarray Analysis of Ovarian Cancer Tissues

Cancer tissues collected from a patient suffering from ovarian cancer during the surgical operation in Samsung Medical Center are frozen with liquid nitrogen and finely crushed in a mortar. Next, Trizol (Invitrogen, Inc.) is introduced into the crushed tissues and the total RNA is separated therefrom according to the instruction by Invitrogen, Inc. After the total RNA is examined quantitatively and qualitatively, it is sent to Genomic Tree Inc. so as to perform microarray analysis thereof with the human 17K cDNA chip (Genomic Tree Inc.). The total RNA extracted from normal tissues of the patient suffering from ovarian cancer is used as a control after pooling the total RNA of normal tissues of each patient or all patients. Expression of various genes varied in the ovarian cancer tissues, and CD9 genes showed an increase in expression throughout the tissues (Fig. 1). Analytical results of fourteen patients suffering from ovarian cancer revealed that CD9 gene expression increased to 4-10 times regardless of the particular type of ovarian cancer (Fig. 2). This demonstrates that CD9 may be used as a target protein for ovarian cancer.

<1-2> Study of CD9 Expression Using Real-Time PCR First, 3 μg of the total RNA extracted from ovarian cancer tissues is used to produce cDNA in the presence of Superscriptase Il Reverse-Transcriptase (invitrogen). Next, the cDNA is diluted with sterilized distilled water to obtain 2% cDNA, which, in turn, is combined with 10 pmole/μL of a CD9 primer mixture [forward primer: 5'-GGGGATATTCCCACAAGGAT-3¹ (SEQ ID NO. 1); a reverse primer: 5'-GATGGCTTTCAGCGTTTCC-3¹ (SEQ ID NO. 2), iQ™ SYBR Green Supermix (Cat. No BR 170-8880, BIO-RAD)] to obtain 20 μl_ of a final mixture. Then, the resultant mixture is subjected to real-time PCR (iCycler iQ, BIO-RAD) in the 8-Strip tube (BIO-RAD). As a control, GAPDH is subjected to real-time PCR. The PCR program is shown in Table 1 :

[Table 1] Real-Time PCR Program

After carrying out the PCR, it was shown that CD9 gene expression increases in ovarian cancer tissues as compared to normal tissues. Especially, CD9 gene expression increased to 5-12 times in borderline tumor and serous type ovarian cancer (Fig. 3). Microarray analysis results of CD9 expression in the patients providing the RNA coincided with the results of the real-time PCR. <1-3> Study of CD9 Expression through Reverse Transcription-

PCR

PCR is carried out by using 2 μl_ of cDNA prepared from 3 μg of the total

RNA, EF-Taq DNA polymerase (SolGent Co., Ltd., Korea) and a primer mixture [forward primer: S'-ATGCCGGTCAAAGGAGGC-S' (SEQ ID NO. 3); reverse primer: 5'-CTAGACCATCTCGCGGTTCC-3' (SEQ ID NO. 4)]. GAPDH RT- PCR is also carried out as a gel loading control. The PCR program is shown in

Table 2:

[Table 2]

After carrying out the PCR, it was shown that CD9 gene expression increases in ovarian cancer tissues as compared to normal tissues. Such results coincide with the real-time PCR results. Therefore, it can be seen that

CD9 may be used as a target protein for ovarian cancer (Fig. 5).

<Example 2> Investigation of CD9 Protein Expression in Ovarian

Cancer Tissues

<2-1> Study of CD9 Expression through lmmunohistochemistry lmmunohistochemical staining is performed with a CD9 antibody (Clone

MM2/57, Millipore) by using a paraffin-embedded ovarian cancer tissue section, lmmunohistochemical analysis is performed according to a generally known method (Shi, S. R., et a/., J. Histochem. Cytochem. 39:741-748, 1991 ; Hori, H., et a/., J. Surg. Res. 117:208-215, 2004). The CD9 antibody is used after being diluted with 1 % BSA/1X PBS at a ratio of 1/50. As a secondary antibody, a biotinylated anti-mouse secondary antibody (1/500 dilution in 1 % BSA/1X PBS, DAKO) is used. Avidin-conjugated HRP (1/500 dilution in 1% BSA/1X PBS, DAKO) is used to perform detection, and the results are observed by using a microscope at 600X.

After the analysis, it was shown that CD9 protein is overexpressed in borderline tumor, serous type ovarian cancer and mucinous type ovarian cancer. In addition, CD9 was expressed well in the ovarian cancer cell membrane. These results conform to the location of CD9 in normal cells known to date in the related art (see Fig. 6).

<2-2> Study of CD9 Expression through Western Blot Analysis Normal tissues and ovarian cancer tissues collected during the surgery are frozen with liquid nitrogen and finely crushed in a mortar. The crushed tissues are transferred to E-tube, and 1 ml_ of lysis buffer A [20 mM Hepes, pH 7.5, 150 mM NaCI, 5 mM MgCI₂, 0.2% Triton X-100, 1 mM PMSF and 1 table/10 mL of complete mini protease inhibitor cocktail] is added thereto before subjecting the tissues to vortex. Then, centrifuge is carried out at 4 ⁰C for 10 minutes, the supernatant is transferred to another E-tube, and a portion of the tissue lysate is collected to perform protein determination. After that, 100 μg of the protein is heated with Laemli buffer free from a reducing agent for 5 minutes, and electrophoresis is carried out by using 12% SDS-PAGE gel. Then, western blot analysis is performed in a generally known manner (Berditchevski, F. and Odintsova, E. J. Cell Biol. 146:477-492, 1999). To detect CD9, 1/1000 dilution of monoclonal antibody (Clone ALB6, Beckman coulter) in PBST (1X PBS and 0.05% Tween 20) is used. As a secondary antibody, 1/5000 dilution of HRP-conjugated anti-mouse IgG (code: NA931 , Amersham) in PBST buffer is used. As a protein loading control, beta(β)-actin level is determined.

After the analysis, it was shown that CD9 protein is overexpressed in serous type ovarian cancer as compared to normal tissues or mucinous type ovarian cancer. In the case of mucinous type ovarian cancer, the result does not conform to the result of CD9 gene expression because of the sensitivity of the particular test method. In other words, the test method for determining gene expression is more sensitive than the western blot analysis, and the stability and turnover time of the protein itself may affect the protein expression (Fig. 7). <2-3> Study of CD9 Protein Expression in Cultured Cancer Cell Line

Ovarian cancer cell lines, cell lines 2774 (Liu, J. R., et a/., Cancer Res. 62:924-931 , 2002; Wu Q., et a/., J. Biol. Chem. 277:36329-36337, 2002; Carroll J. L., et ai, in Methods in Molecular Medicine, Vol. 39:783-792 Humana Press: Totowa, NJ. USA) available from ATCC (American Type Cell Collection), Ovcar3 (HTB-161) and SKOV3 (HTB-77), cervical cancer cell line HeLa (CCL- 2), breast cancer cell line MCF-7 (HTB-22), and HEK 293 (human embryonic kidney 293; CRL-1573) cells are cultured in 100 mm dishes, and washed with 1X cold PBS twice when the cells are filled to about 90%. Next, lysis buffer A is added thereto, and reaction is carried out for 1 hour at 4 ⁰C. The cells are scraped and transferred to E-tube. Then, the cells are subjected to centrifuge at 4 ⁰C for 10 minutes and the supernatant is transferred to another E-tube to perform protein determination. After that, 50 μg of protein from each cell line is subjected to electrophoresis using 12% SDS-PAGE gel, and western blot analysis is performed. As a control of the amount of protein, the amount of alpha (α)-tubulin is measured by using the same nitrocellulose membrane. After the analysis, it was shown that CD9 protein was the most highly expressed in cell line 2774 among the ovarian cell lines, and CD9 was less expressed in SKOV3 than the normal cell line, HEK 293. Additionally, CD9 was highly expressed also in the cervical cancer cell line, HeLa, and the breast cancer cell line, MCF-7 (Fig. 8).

<Example 3> Investigation of Location of CD9 Expression Using Immunofluorescence Microscopy

Immunofluorescence microscopy is performed at room temperature. All the solutions used therein are prepared by using 1X PBS. Ovarian cancer cell line 2774 that highly expresses CD9 is selected to perform the experiment in this example.

A 6-well plate is covered with a cover glass, and coated with gelatin, and then 2774 ovarian cancer cell line is cultured. 36 hours after the culture, the cell line is fixed with 4% formaldehyde for 10 minutes, and membrane permeation is performed with 0.05% Triton X-100 for 5 minutes. After the cells are blocked with 3% BSA for 30 minutes, staining is performed for 1.5 hours with a CD9 antibody (ALB6) diluted with dilution buffer (0.1% BSA, 0.05% Triton X-100 in PBS) to 1/200. Next, the cells are washed with 1X PBS three times and staining is further performed for 45 minutes with a secondary antibody (anti- mouse Alexa 568, Molecular Probes, Cat. No. A11019) diluted with dilution buffer to 1/2500. The cells are washed with 1X PBS three times, stained with DAPI (100 ng/mL) for 5 minutes and further washed with PBS. The cover glass is mounted on a glass slide and sealed with nail polish.

After the experiment, it was shown that CD9 exists in the cell membrane of 2774 cell line. This conforms to the results of immunohistochemical analysis. Therefore, it can be seen that the location of CD9 expression in cancer cells shows no change as compared to the location of CD9 expression in normal cells.

<Example 4> Study of CD9 functions

<4-1> Study of Growth of CD9/SKOV3 Cell Line

Flag-tagged CD9 is subjected to stable transfection into SKOV3 cell line to produce CD9/SKOV3 cell line that stably overexpresses CD9. Screening of CD9/SKOV3 stable cells is performed through western blot using Flag antibodies. SKOV3 (Vec/SKOV3), only the flag vector of which is stably transfected, and the other two clones (clones 14 and 16) of CD9 are selected and cultured on a 96-well plate (6 wells for each sample, 6000 cells per well). microculture tetrazolium (MTT) analysis is performed, 24, 48, 72, 96 and 120 hours after the culture. MTT analysis is carried out as follows. To the cells from which the culture medium is removed, MTT solution (5 mg/mL in PBS; MTT, Sigma Cat. No. M2128) sterilization-filtered through a 0.45 μm membrane filter is added in an amount of 50 μL/well. After culturing at 37 ⁰C for 4 hours, MTT solution is removed, DMSO is added in an amount of 50 μL/well, and then absorbance is measured on a microplate reader (560 nm). The absorbance value represents the living cell count.

As a result, it was shown that CD9 stable cell lines (both clone Nos. 14 and 16) stimulated cell growth as compared to Vec/SKOV3. This demonstrates that CD9 stimulates cell growth in SKOV3 cells (Fig. 10 or 11 ).

<4-2> Study of Cell Growth in Ovarian Cancer Cell Line Using CD9 Monoclonal Antibody

ALB6 (Beckman Couter Inc.), a known CD9 monoclonal antibody, is added to 2774 cells to investigate cell growth behavior when endogenous CD9 is blocked. Cell growth is determined in real time by using RT-CES (real time- cell electronic sensing system, ACES Biosciences, Inc.). The system reads the count of cells adhering to a plate by detecting the resistance to the current generated upon the adhesion of the cells to the plate. Next, 2774 cells are loaded on a 96-well plate (5000 cells per well), and ALB6 as a CD9 antibody and control mouse IgGI are added in an amount of 50 μg/mL. Then, the plate is introduced into the RT-CES system placed in an incubator under the conditions of 37 ⁰C, 5% CO₂. The cell growth is determined with one hour interval in real time, and the determination is continued for 90 hours (about 3 days). The cell count at 75 hours after the antibody treatment and the cell count in the ALB6-treated group are represented by a bar graph, relative to the cell count of the control (Fig. 12).

As a result, it was shown that cell growth was inhibited in the CD9 antibody-treated group as compared to the PBS- or control IgGI -treated groups. This suggests that inhibition of CD9 functions caused by the CD9 antibody results in inhibition of cell growth. Therefore, it can be seen that CD9 stimulates cell growth in SKOV3 and 2774 cell line.

<4-3> Study of Effect of CD9 Monoclonal Antibody upon Invasion and Migration of Ovarian Cancer Cell Line

To perform invasion assay, 0.6 mL/well of a culture medium, to which 0.1 % BSA is added, is loaded to a 24-well plate, a transwell coated with Matrigel (at the inner part of the transwell filter) and collagen (at the outer part of the transwell filter) is introduced into each well, and 0.4 mL/well of the medium is further added to a final volume of 1 mL/well. Next, 2774 cell line is treated with trypsin/EDTA and is subjected to centrifuge. The cells precipitated after the centrifuge is resuspended with PBS containing 0.1% BSA. Then, 5X10³ cells of 2774 cell line are introduced into each well, IgGI and ALB6 antibodies are added thereto, each in an amount of 50 μg/mL, and the cells are cultured overnight in a cell culture incubator at 37 ⁰C. After the culture, the transwell is fixed with 100% methanol and stained with hematoxylin and eosin. The transwell membrane is stripped, loaded on a slide glass, covered with a cover glass, and sealed with nail polish. After that, the invaded cell count is determined by counting the cells by microscopy. Migration assay is also performed in the same manner as the invasion assay, except that the transwell used in the assay is not coated with Matrigel.

As a result, it was shown that ALB6 inhibits cell invasion to about 25% and cell migration to about 50%, as compared to the control IgGI . Thus, it can be seen that CD9 stimulates invasion and migration in 2774 cell line (Fig. 13 or 14). <Example 5> Study of CD9-Related Signaling Pathway

In this example, CD9-related signaling pathways are studied. First, microarray analysis of CD9/SKOV3 stable cell line is performed to investigate genes up-regulated by overexpression of CD9 as compared to Vec/SKOV3. CD9/SKOV3 stable cell line No. 14 clone and No. 16 clone are cultured in a 100 mm dish until the cells are filled to about 90%. The cells are washed with 1X cold PBS twice, Trizol is added thereto in an amount of 1 ml_ per dish, and the total RNA is extracted. After measuring the concentration of the total RNA, it is sent to Genomic Tree Inc. so as to perform microarray analysis.

As a result, it was shown that CD9 overexpression significantly controls expression of a wide variety of genes related to NF-κB signaling pathway. Typically, NF-κB signaling pathway plays an important role in tumorigenesis of cells by inducing gene transcription required for cell growth through the migration of NF-κB toward the nucleus, when NF-κB in cells is activated due to the binding of TNF-α to TNFR-α receptor. Overexpression of CD9 results in an increase in expression of genes required for the activation of NF-κB, and target genes of NF-κB increase accordingly. Therefore, it can be seen that CD9 activates NF-κB signaling pathway and stimulates tumorigenesis. [Table 3]

CD9/SKOV3 Microarray Data Related to NF-κB Signaling Pathway

In addition, Vec/SKOV3 and CD9/SKOV3 cell lines are subjected to subcellular fractionation and the migration of p65, a subunit of NF-κB, toward the nucleus is monitored via western blot analysis. After the analysis, it was shown that p65 migrates highly toward the nucleus due to the overexpression of CD9. This demonstrates that CD9 activates NF-κB signaling pathway (Fig. 15). Further, a 6-well plate is covered with a cover glass and coated with gelatin to culture Vec/SKOV3 and CD9/SKOV3 cell lines. Then, the location of p65 in cells is determined by immunofluorescence microscopy. As a result, it was shown that p65 migrates highly toward the nucleus in CD9/SKOV3 cell line. This also demonstrates that CD9 activates NF-κB signaling pathway (Fig. 16).

<Example 6> Study of CD9 Functions in Ovarian Cancer Animal

Model An ovarian cancer animal model is established by injecting ovarian cancer 2774 cell line that stably expresses green fluorescence protein intraperitoneal^ to 7 week-aged female nude mice (BALB/C nu/nu) in an amount of 5X10⁶ cells per mouse (Furuya, M., et al., Cancer Res. 65:2617-2625, 2005; Zvieriev, V., et al., Biochem. Biophys. Res. Commun. 337:498-504, 2005). One week after the injection of 2774 cells, ALB6, a CD9 antibody, is injected intraperitoneal^ at a dose of 2 mg per kg of mouse body weight with 3-7 day intervals. Mouse antibody (IgGI) is injected as a control. After injecting the antibodies four to five times, the mice are sacrificed and cancer generated in the abdominal cavities is investigated.

As a result, it was shown that the weight of tumor is decreased by inhibiting CD9 functions (Fig. 17). The weight measurement results of tumors are shown as a bar graph (Fig. 18). In addition, to confirm the inhibition of cancer cell growth caused by the adhesion of ALB6 CD9 antibodies to the cancer in the abdominal cavities of mice, cancer cells of the mice, to which IgGI and ALB6 antibodies are injected, are subjected to immunohistochemical staining using mouse secondary antibodies. After the experiment, it was shown that only the mouse tumors, to which ALB6 was injected, were stained (Fig. 19). Therefore, it can be seen that the decrease in the weight of mouse

tumor, to which ALB6 was injected, results from the fact that ALB6 inhibits the

functions of CD9.

[Sequence List Text]

See the attached file for the sequence list (C:\KipoNet\KEditor\appfile\8p-01-24.app).

While the exemplary embodiments have been shown and described, it

will be understood by those skilled in the art that various changes in form and

details may be made thereto without departing from the spirit and scope of this

disclosure as defined by the appended claims.

In addition, many modifications can be made to adapt a particular

situation or material to the teachings of this disclosure without departing from

the essential scope thereof. Therefore, it is intended that this disclosure not be

limited to the particular exemplary embodiments disclosed as the best mode

contemplated for carrying out this disclosure, but that this disclosure will include

all embodiments falling within the scope of the appended claims.

Claims

[CLAIMS] [Claim 1 ]

An anticancer agent comprising a CD9 inhibitor as an active ingredient.

[Claim 2]

The anticancer agent according to claim 1 , wherein the CD9 inhibitor inhibits growth of cancer cells.

[Claim 3]

The anticancer agent according to claim 1 , wherein the CD9 inhibitor inhibits invasion of cancer cells.

[Claim 4]

The anticancer agent according to claim 1 , wherein the CD9 inhibitor inhibits migration of cancer cells.

[Claim 5] The anticancer agent according to any one of claims 1 to 4, wherein the CD9 inhibitor is selected from the group consisting of antisense nucleotides that bind complementarily to mRNA of CD9, siRNA molecules, substrate analogues and antibodies that bind to CD9 and small compounds inhibiting expression of CD9.

[Claim 6]

The anticancer agent according to any one of claims 1 to 4, wherein the cancer is solid cancer that overexpresses CD9.

[Claim 7]

The anticancer agent according to claim 6, wherein the cancer is ovarian cancer.

[Claim 8]

A method for treating cancer, which comprises administering a pharmaceutically effective amount of the anticancer agent as defined in claim 1 to a subject suffering from cancer. ICIaim 9]

The method according to claim 8, wherein the cancer is solid cancer that overexpresses CD9.

[Claim 10]

The anticancer agent according to claim 9, wherein the cancer is ovarian cancer.

[Claim 11 ]

A method for tracing cancer, which comprises: administering an antibody that binds specifically to CD9 to a subject; and imaging locations of the antibody.

[Claim 12]

The method according to claim 11 , wherein the antibody is a polyclonal antibody, a monoclonal antibody or a fragment thereof. [Claim 13]

The method according to claim 11 , wherein the imaging is carried out through fluorescence, luminescence, chemiluminescence, absorbance, reflection or transmission by using a conjugate labeled with a chromophohc enzyme, fluorescent material, radioactive isotope or colloid.

[Claim 14]

The method according to claim 11 , wherein the cancer is solid cancer that overexpresses CD9.

[Claim 15]

The method according to claim 14, wherein the cancer is ovarian cancer.

[Claim 16]

A kit for diagnosing cancer, which comprises an antibody that binds specifically to CD9.

[Claim 17] The kit according to claim 16, which is based on determination using surface plasmon resonance (SPR) systems or surface plasmon resonance imaging (SPRI) systems; or fluorescence spectroscopy including detection of fluorescence caused by a fluorescent marker attached to an antibody.

[Claim 18]

The kit according to claim 16, wherein the cancer is solid cancer that overexpresses CD9.

[Claim 19]

The kit according to claim 18, wherein the cancer is ovarian cancer.

[Claim 20]

A method for diagnosing cancer, which comprises: measuring an amount of CD9 expression in cancer candidate tissue-derived cells; and comparing the amount of CD9 expression with an amount of CD9 expression in normal tissue-derived cells to select tissues in which CD9 expression is increased. [Claim 21 ]

The method according to claim 20, wherein the amount of CD9 expression in cancer candidate tissue-derived cells is determined by microarray, real-time PCR and reverse transcription PCR using genes, and immunohistochemical analysis and western blot analysis using proteins.

[Claim 22]

The method according to claim 20, wherein the cancer is solid cancer that overexpresses CD9.

[Claim 23]

The method according to claim 22, wherein the cancer is ovarian cancer.