WO2007114550A1 - A novel monoclonal antibody specific to the l1cam, a hybridoma producing the same and a method producing the same - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically binds to LlCAM protein. The novel antibody A10-A3 according to the present invention can specifically bind to the LlCAM protein, which is expressed in various cancers including cholangiocarcinoma and lung cancer, so as to suppress the growth, invasion, and migration of the cancer cells.

Description

A NOVEL MONOCLONAL ANTIBODY SPECIFIC TO THE LlCAM, A HYBRIDOMA PRODUCING THE SAME AND A METHOD PRODUCING THE SAME

[Technical Field] The present invention relates to a monoclonal antibody that specifically binds to LlCAM, a hybridoma producing the same, and a method for producing the same. Particularly, the present invention relates to a novel monoclonal antibody obtained from the abdominal cavity of a mouse immunized with a cholangiocarcinoma cell lines, which specifically binds to LlCAM protein expressed in various cancer cells such as cholangiocarcinoma and lung cancer, a hybridoma producing the same, and a method for producing the same.

[Background Art] LlCAM (Ll cell adhesion molecule) is one of integral membrane glycoproteins belonging to a large class of immunoglobulin superfamily cell adhesion molecules (CAMs) that mediate cell-to-cell adhesion at the cell surface, and its molecular weight is up to 220 kDa. LlCAM is found primarily in neurons, hematopoietic cells, renal cells, or the like (Bateman, et al., EMBO J. 15:6050-6059; 1996) and plays a role in neuronal migration, neurite outgrowth, and cell migration. A human LlCAM gene is isolated from a human fetal brain cDNA library using degenerate oligonucleotides as probes in mouse and rat LlCAM homologs (Hlavin, M. L. & Lemmon, V. Genomics 11: 416-423, 1991; U.S. Patent No. 5872225, granted on Feb. 16, 1999). It is known that LlCAM is mainly expressed in brain. However, it has been found in some normal tissues, and recently in various cancer cells.

With respect to correlation between LlCAM and cancer, it has been reported that LlCAM is expressed in various cancers including melanomas, neuroblastoma, ovarian carcinoma, and colon cancer (Takeda, et al., J. Neurochem. 66:2338-2349, 1996; Thies et al, Eur. J. Cancer, 38:1708-1716, 2002; ArIt et al . , Cancer Res. 66:936-943, 2006; Gavert et al., J. Cell Biol. 168:633-642, 2005), and its cleavage product was found to be secreted out of the cell, as well as its membrane-bound form (Gutwein et al . , FASEP J. 17(2):292-4, 2003). Further, LlCAM was recently identified as one of molecules playing an important role in tumor growth (Primiano, et al., Cancer Cell. 4(l):41-53, 2003), and has become a new target for cancer treatment (U.S. Patent Application Publication No. 2004/0115206 Al, filed on June, 17, 2004). Recently, it was also found that LlCAM is expressed in the invasive front of colon cancer (Gavert, et al., J Cell Biol. 14; 168(4) :633-42, 2005), and an antibody against LlCAM inhibits tumor cell growth and metastasis in ovarian carcinoma (ArIt, et al . , Cancer Res. 66:936-943, 2006). On the other hand, U.S. Patent Application No. 2004/0115206 discloses that the effects of LlCAM antibodies were reported to stimulate rather than inhibit the effects of LlCAM on signal transduction. Thus, LlCAM antibodies do not necessarily inhibit the effects of LlCAM. EP 1 172 654 Al, and U.S. Patent No. 2004/0259084 disclose a method for a diagnosis or a prognosis of an ovarian or endometrial tumor, in which the LlCAM level is determined by the anti-LlCAM antibody in a patient sample, based on the presence of LlCAM being an indication of the presence of an ovarian or endometrial tumor or a possible predisposition for such a tumor, and a method for treating cancer which comprises administering to the patient a sufficient amount of an anti-LICAM antibody conjugated to a cytotoxic drug. Further, U.S. Patent Application No. 2004/0115206 discloses a reagent for inducing tumor cell death using antibodies that specifically bind to LlCAM, a method of using the antibodies for inducing cell death, and a pharmaceutical composition comprising the anti-LICAM antibodies, in which a method for inhibiting growth or inducing cell death in a tumor cell by contacting the tumor cell with an effective amount of an anti-LICAM antibody to inhibit cell growth or induce cell death in the tumor cell is disclosed. Further, International Patent Application PCT/EP2005/008148 provides a composition inhibiting the LlCAM protein being overexpressed in ovarian and endometrial carcinoma and its expression, and a method for preventing and treating ovarian and endometrial carcinoma using the composition. The document describes that a composition comprising an anti-LICAM antibody inhibiting the LlCAM function and its derivative inhibit the function of ovarian and endometrial carcinoma cell, so as to suppress migration of cancer cells and progression of cancer, thereby treating cancer.

However, the documents do not describe that a novel antibody A10-A3 and an antibody specifically binding to the IgI domain of LlCAM effectively suppress the growth, migration, and invasion of various tumor cells including ovarian carcinoma, breast carcinoma, cholangiocarcinoma and lung cancer, and they are antibodies to induce tumor cell death. The present inventors have made extensive studies on developing an antibody which can be used for diagnosing and treating cholangiocarcinoma. As a result, the inventors obtained a monoclonal antibody that specifically binds to the surface of cholangiocarcinoma by immunizing mice with a cholangiocarcinoma cell line, which was recently established (Kim et al, Genes, chromosome & Cancer 30:48-56, 2001), and they designated the antibody as an A10-A3 antibody. In addition, the present inventors found that the A10-A3 antibody specifically binds to the LlCAM protein. They also found that the A10-A3 antibody specifically binds to tumor cells such as breast carcinoma, ovarian carcinoma, lung cancer, colon cancer, and skin cancer, as well as cholangiocarcinoma, but the A10-A3 antibody does not bind to normal cells such as peripheral blood lymphocytes, liver cells, and blood endothelial cells. Until now, LlCAM has been known to be expressed in breast carcinoma, ovarian carcinoma, colon cancer, skin cancer, or the like, but LlCAM has not been known to be expressed in cholangiocarcinoma or lung cancer. Therefore, the present inventors examined whether the LlCAM antibody suppresses the growth or migration of cholangiocarcinoma or lung cancer. As a result, they found that the A10-A3 antibody has a significant effect of suppressing the growth, migration, and invasion of tumor cells such as cholangiocarcinoma and lung cancer.

In order to confirm whether the A10-A3 antibody of the present invention is a novel monoclonal antibody being specific to LlCAM, the present inventors analyzed the epitopes of the A10-A3 antibody, and compared them with those of 5G3 and UJ127 which are the known monoclonal antibodies against LlCAM. Consequently, it was confirmed that the A10-A3 antibody binds to the IgI domain of LlCAM, while 5G3 and UJ127 bind to an Ig2 domain and a fibronectin type III domain, respectively. Thus, A10-A3 was found to be a novel monoclonal antibody against LlCAM.

Therefore, the present inventors confirmed that the novel A10-A3 antibody according to the present invention specifically binds to the IgI domain of the LlCAM protein to effectively suppress the growth, migration, and invasion of tumor cells, and it is an antibody to induce tumor cell death, thereby completing the present invention.

[Disclosure] [Technical Solution]

It is an object of the present invention to provide a novel monoclonal antibody that specifically binds to LlCAM, a hybridoma producing and secreting the monoclonal antibody, and a method for mass-producing the monoclonal antibody by culturing the hybridoma.

It is another object of the present invention to provide a method for inhibiting growth and invasion of cancers including cholangiocarcinoma and lung cancer, in which LlCAM is expressed, and a pharmaceutical composition comprising the said antibody.

[Description of Drawings] Fig. 1 is a graph showing whether a murine monoclonal antibody A10-A3 (Fig. IA), a known antibody 5G3 (Fig. 1C) and UJ127 (Fig. IB) bind to the surface of various tumor cells including cholangiocarcinoma and small cell lung cancer, and the surface of normal cells, using fluorescence staining and a flow cytometer; Fig. 2 is a photograph showing the result that the antigen, which the A10-A3 antibody binds to, is LlCAM protein, by immunoprecipitation and Western blotting;

Fig. 2A is a photograph showing the result of biotinylating the surfaces of cholangiocarcinoma cells (Choi-CK cells), immunoprecipitating with the A10-A3 antibody or the known anti-LlCAM monoclonal antibody (UJ127), and then performing Western blotting with the precipitated protein using 10% SDS-PAGE and Streptavidin-HRP, and Fig. 2B is a photograph showing the result that Western blotting is performed with the protein, which is immunoprecipitated with the AlO- A3 antibody, using the known anti-LICAM antibody (UJ127) on the 10% SDS-PAGE, so as to detect LlCAM, in which preclearing represents a negative control being immunoprecipitated without any antibody, IP with A10-A3 represents those immunoprecipitated with the A10-A3 antibody, IP with anti-LICAM represents those immunoprecipitated with the known monoclonal antibody against LlCAM, and A10-A3 only represents the antibody only loaded on the SDS-PAGE;

Fig. 3 is a diagram showing that the protein, which is immunoprecipitated with the A10-A3 antibody in the Choi-CK cells, is separated from SDS-PAGE, and cleaved by trypsin, and the obtained peptide is confirmed to be LlCAM by a Q-TOF analysis, in which the lower amino acid sequence represents the entire LlCAM, and the upper amino acid sequence is an amino acid sequence of the analyzed peptide and corresponds to the underlined part in the entire LlCAM; Fig. 4 is a photograph showing the result of the domain of LlCAM recognized by the A10-A3 antibody; Fig. 4A is a photograph showing the result of performing Western blotting for an Ig dom protein or an Fn dom protein, which is produced by fusing an immunoglobulin (Ig) domain or a fibronectin type III (Fn) domain of the LlCAM with a human Ig Fc, using the A10-A3, 5G3, and UJ127 antibody respectively, and Fig. 4B is a photograph showing the result of Western blotting for the proteins, which are produced by fusing an Ig Fc with water-soluble LlCAM (SoILl), Igl-6, Igl-5, Igl-4, Igl-3, Igl-2, IgI, and Ig2-6 domain among Ig domains, respectively, using the A10-A3 antibody, and Fig. 4C is a photograph showing the result of performing Western blotting for the Ig variants using the 5G3 antibody;

Fig. 5 is a photograph showing the result of immunohistochemical staining of tissues from patients with cancers using the A10-A3 antibody, in which the antibody does not bind to normal liver tissue, and binds to the tissue of cholangiocarcinoma, breast carcinoma, ovarian carcinoma, skin cancer, pancreatic cancer, small cell lung cancer, and non-small cell lung cancer in the body; Fig. 6 is a graph showing whether the growth of cholangiocarcinoma cell line Choi-CK and small cell lung cancer cell line DMS114 is suppressed by the A10-A3 antibody (Fig. 6A) or the known antibody UJ127 or 5G3 against LlCAM (Fig. 6B). 10 μg/ml of the antibody is added to the cells under culturing to culture for 72 hours, and then the growth inhibition is expressed as percentage to the control, to which the antibody is not added. As a positive control of the cell, ovarian carcinoma cell line SK-0V3 is used, and as a negative control of the cell, renal carcinoma cell line ACHN that does not bind with the A10-A3 antibody is used. Besides, as a negative control of the antibody, the antibody is not added, or inactivated antibody (A10-A3b) by heating, or a normal mouse IgG is used; Fig. 7 is a graph showing whether the invasion and migration of cholangiocarcinoma cell line (Choi-CK, SCK), small cell lung cancer cell line DMS114, and ovarian carcinoma cell line (SK-0V3) are suppressed by the A10-A3 antibody. 10 μg/ml of the antibody is added to the cells under culturing to culture for 72 hours, and then the invasion (Fig. 7A) and migration (Fig. 7B) of the cells are expressed as percentages to the control, to which the antibody is not added. As a negative control of the cell, renal carcinoma cell line ACHN that does not bind with the A10-A3 antibody is used, and as a negative control of the antibody, the antibody is not added, or a normal mouse IgG is used; Fig. 8 is a photograph showing the effect of inducing the cell death of cholangiocarcinoma by the A10-A3 antibody, in which a negative control is treated with mouse immunoglobulin IgG,

Fig. 8A is a photograph showing the effect of inducing the cell death by the A10-A3 antibody, in which each cell was treated with the A10-A3 antibody, and then reacted with Annexin-V-FITC to confirm its binding to the cell surface, and

Fig. 8B is a photograph showing the FACS results of detecting the Caspase-3 activated by the A10-A3 antibody; Fig. 9 is a photograph showing the result of analyzing cell signal transduction to confirm the inhibitory effect of the A10-A3 antibody on the growth, invasion, and migration of the cancer cells, in which cholangiocarcinoma cell line, Choi-CK or SCK was cultured in the medium containing no antibody, or in the medium containing the A10-A3 antibody or mouse immunoglobulin IgG, then the collected cell extracts was analyzed with an antibody against β-actin, and Western blotting was performed using antibodies against PCNA (Fig. 9A), phospho-MAPK (Fig.9A), phospho-AKT (Fig. 9B), and phospho-FAK (Fig. 9C);

Fig. 10 is a photograph showing the FACS results that confirm integrin expression in the cancer cells (Fig. 10A), and a graph showing the results that the inhibitory effect of an antibody against integrin and the A10-A3 antibody on the cancer cell growth were compared (Fig. 10B);

Fig. 11 is a graph showing the results that the A10-A3 antibody suppresses the cell growth of cholangiocarcinoma induced by the soluble LlCAM, in which ♦ represents the result of observing the cell proliferation in the case of adding the soluble LlCAM with various concentrations to the culture media for cholangiocarcinoma cells, and ■ represents the result of observing the cell proliferation in the case of adding a mixture of the soluble LlCAM and the A10-A3 antibody to the culture media for cholangiocarcinoma cells,

Fig. HA is the results obtained by using the Choi-CK cells, and Fig. HB is the results obtained by using the SCK cells; and Fig. 12 is a graph showing the experimental result that demonstrates the inhibitory effect of the A10-A3 antibody on cancer cell growth in a xenografted mouse model of human cholangiocarcinoma,

Fig. 12A is the result of analyzing the tumor sizes of five mice injected with the antibody (A10-A3 group) and five mice injected with no antibody (control group) over time, Fig. 12B is the result of measuring the tumor weight at 3 weeks after tumor implantation, and

Fig. 12C is the result of measuring the body weight.

[Best Mode] The present invention relates to a novel monoclonal antibody that specifically recognizes LlCAM protein.

The term 'Monoclonal antibody'?as used herein, means a highly specific antibody which is directed against a single antigenic site (epitope), as known in the art. Typically, unlike a polyclonal antibody including different antibodies directed against different epitopes, a monoclonal antibody is directed against a single epitope on an antigen. The monoclonal antibody has an advantage that selectivity and specificity are improved in diagnosis and analysis using the antigen- antibody binding. In addition, the monoclonal antibody has another advantage that it is produced by the culturing of hybridoma to avoid contamination with other immunoglobulins.

The monoclonal antibody can be produced by a fusion method well known in the art (Kohler et al., European Journal of Immunology 6; 511-519). Generally, the hybridoma secreting the monoclonal antibody is prepared by fusing an immune cell, which is from an immunologically acceptable host animal such as a mouse injected with an antigen protein, with a cancer cell line. The cell fusion between two cells is induced by a method well known in the art to which the present invention pertains, such as polyethylene glycol, and the antibody-producing cells are amplified by a standard culture method. The cells are subcloned by limited dilution to obtain a uniform cell population, and then a hybridoma cell producing an antibody specific to an antigen is mass- produced in vitro or in vivo.

The monoclonal antibody of the present invention can be used without purification, or alternatively used after high-purification by a variety of usual methods such as dialysis, salt precipitation, ion exchange chromatography, size exclusion chromatography, and affinity chromatography.

In the specific examples of the present invention, the monoclonal antibody is secreted by the hybridoma according to the present invention, and specifically binds to the cell surface of cholangiocarcinoma or lung cancer. Further, the monoclonal antibody binds to other cancer cells including liver cancer, ovarian cancer, gastric cancer, skin cancer, colon cancer, and breast cancer, in addition to the said cancer cells (see Fig. 1), but does not bind to normal cells such as liver cells, HUVEC (human-derived umbilical vein endothelial cells), and peripheral blood lymphocyte (see Fig. 1). The monoclonal antibody binds to an antigen to suppress or neutralize its actions, or even kill the cells. For example, the monoclonal antibody which specifically recognizes cancer cells can bind to the cell surface molecules, or the water-soluble molecules, which are involved in the growth or migration of cancer cells, so as to suppress or neutralize their action. Further, the antibody binding to the antigen can activate a complement, and allow the activated complement to remove the antigen. In addition, the monoclonal antibody binds to the antigen, and may promote the antigen to be more easily ingested by the phagocytic cells. The antigen bound with the monoclonal antibody can be easily killed by a natural killer cell (NK cell), and thus the monoclonal antibody can be used to remove the antigen through an immune response. Therefore, it can be expected that the antibody is used alone to remove or reduce the antigen through an immune response, whereby the antibody of the present invention can be used for diagnosis or treatment of various cancers including cholangiocarcinoma and lung cancer. In a specific embodiment of the present invention, the monoclonal antibody binds to cancer cells so as to suppress their growth or migration, or so as to ingest, suicide, or kill the cancer cells. Specifically, the monoclonal antibody can bind to LlCAM that is a surface antigen of the cancer cell, and suppress the activity of LlCAM, thereby inhibiting the growth and metastasis of the cancer cells. More specifically, the monoclonal antibody binds to an IgI domain of LlCAM, and thus it can be used for diagnosis or treatment of cancers expressing LlCAM.

In another specific embodiment of the present invention, the monoclonal antibody binds to the secreted LlCAM to inhibit the growth of cancer cells (see Example 11).

U.S. Patent Application Publication No. 2004/0115206 describes that UJ127 and 5G3, which are two commercial antibodies against LlCAM, are used to induce the cell death in the cell lines of breast carcinoma, colon cancer, or cervical cancer. However, in this document, as shown in Examples of the present invention, when the cells of cholangiocarcinoma and small cell lung cancer are treated with these known antibodies specific to LlCAM, the 5G3 antibody binds to cholangiocarcinoma and small cell lung cancer, but do not suppress any growth of the small cell lung cancer. Meanwhile, the monoclonal antibody according to the present invention has a remarkable effect that it can bind to various cancers such as cholangiocarcinoma or lung cancer, as well as suppress their growth, invasion, and migration. This is believed to occur because the monoclonal antibody specific to LlCAM according to the invention has different epitopes binding to LlCAM and affinity from 5G3, as described in detail in Examples below. On the other hand, in still another embodiment of the present invention, a well known therapeutic agent can bind to the monoclonal antibody directly or indirectly to provide a more effective cancer treating substance. Examples of the therapeutic agent capable of binding to the antibody include radionuclides, drugs, lymphokines, toxins, and bispecific antibodies.

Examples of the radionuclide include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re, but are not limited thereto. Examples of the drug and toxin include etoposide, teniposide, adriamycin, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, cis-platinum and cis-platinum analogues, bleomycins, esperamicins, 5-fluorouracil , melphalan, and other nitrogen mustards, but are not limited thereto. It will be apparent to those skilled in the art that the monoclonal antibody according to the present invention can be converted into a chimeric antibody, a humanized antibody, and a human monoclonal antibody, of which immunogenicity is reduced for applications in human bodies. The chimeric antibody is produced by recombination of a variable region of the monoclonal antibody of the invention and a constant region of a human antibody, the humanized antibody is produced by implanting complementarity determining regions (CDRs), which directly bind to an antigen, in a variable region of the monoclonal antibody of the present invention, or specificity determining residues (SDRs), which are involved in antigen binding specificity among the complementarity determining regions, into a human antibody.

The human antibody can be easily produced from the monoclonal antibody of the present invention using a well known method that comprises the steps of replacing a heavy-chain variable region, or a light-chain variable region among the variable regions of the monoclonal antibody of the present invention with a heavy-chain variable region, or a light-chain variable region of a human antibody, then replacing a mouse heavy-chain variable region, or a mouse light-chain variable region from the resulting hybrid (mouse heavy-chain/human light-chain, or mouse light-chain/human heavy-chain) antibody having antigen- binding capacity, selecting a complete human antibody variable region having antigen-binding capacity, and linking it with the constant region of the human antibody. It is a matter of fact that these variants are encompassed by the present invention. Subsequently, the chimeric antibody, the humanized antibody, and the human monoclonal antibody can be produced in animal cells using a well known method. Further, as long as the monoclonal antibody of the present invention has the binding property as described above, it is a complete form having a full length of two heavy-chains and two light-chains, as well as a functional fragment of the antibody molecule, and thus it can be used for cancer treatment and diagnosis. Here, the phrase "the functional fragment of the antibody molecule" means a fragment containing at least antigen-binding capacity, and may include Fab, F(ab'), F(ab')₂, and Fv. Further, the present invention relates to a hybridoma producing and secreting the monoclonal antibody which specifically recognizes a surface antigen of cancer cells, LlCAM.

The term 'Hybridoma' as used herein, means a cell obtained by fusion of an antibody-producing B lymphocyte and a myeloma cell, in which the myeloma cell is a transformed cell and is advantageous in that it can be cultured in a culture plate for a long time. That is, if a myeloma hybridoma producing only one antibody is isolated, the monoclonal antibodies can be easily obtained in a massive amount. The hybridoma of the present invention producing the monoclonal antibody according to the present invention can be prepared by the method, in which the cell expressing LlCAM is mass-cultured, then a mouse is immunized with the cell, and the lymphocyte collected from the immunized mouse is fused with a myeloma cell. Examples of the myeloma cell used in cell fusion may include various cell lines such as mouse-derived p3/x63~Ag8, p3-Ul, NS-I, MPC-Il, SP- 2/0, FO, P3x63Ag8, V653, S194, and rat-derived R210. The cell line used in the specific examples of the invention is myeloma cell FO. Further, the method for producing the hybridoma can further comprise a step of screening a hybridoma which produces a monoclonal antibody specific to cholangiocarcinoma cell. Preferably, using fluorescence cell staining, a mouse hybridoma producing a mouse monoclonal antibody specific to cholangiocarcinoma cell can be screened.

As one specific Example, the present inventors mass-cultured cholangiocarcinoma cells, injected the cells to the paw pad of a mouse, isolated lymphocytes from the lymph nodes of the mouse, and fused them with the myeloma cells, thereby producing a mouse hybridoma which produces an antibody specific to cholangiocarcinoma. More specifically, the present inventors injected the cholangiocarcinoma cells, SCK and Choi-CK (Kim et al . , Genes, chromosome & Cancer 30:48-56, 2001) to the paw pad of a mouse, and isolated lymphocytes from the popliteal lymph nodes of the mouse. The lymphocytes and the FO myeloma cell lines were fused, and clones expressing the antibodies were screened. Among the above-prepared clones, the hybridoma supernatants that stably secrete the monoclonal antibodies were selected and their binding capacity for cholangiocarcinoma and lung cancer cell was examined. The monoclonal antibody and the hybridoma secreting the monoclonal antibody were designated as Hybridoma A10-A3, and deposited in Genbank of Korea Institute of Bioscience and Biotechnology (Deposit No.: KCTC10909BP) on Feb. 20, 2006.

The present invention further relates to a method for mass-producing the monoclonal antibody using the hybridoma.

In one embodiment of the present invention, the method for mass- producing the monoclonal antibody comprises the steps of intraperitoneal Iy injecting the screened hybridoma to a mouse, culturing it in its ascitic fluid, and isolating it by protein G- sepharose column chromatography or the like, but not limited thereto. Further, if a desired hybridoma clone is isolated, this clone is cultured. Thereafter, the monoclonal antibodies in the culture media can be isolated and used, and the monoclonal antibody can be obtained from the ascitic fluid obtained by intraperitoneal Iy injecting the hybridoma clone to a mouse. The prepared monoclonal antibody can be used in wide applications such as immunodiagonsis, immunotherapy, and molecular cell biological studies on antigens and antibodies, and further used in obtaining a useful antibody such as a catalytic antibody. Preferably, the method for mass-producing the monoclonal antibody can further comprise a high purification process (e.g., 95% or more) using a method known in the related art. The monoclonal antibody can be isolated from the culture medium or the ascitic fluid using a purification method such as gel electrophoresis, dialysis, salt precipitation, and chromatography.

Further, the present invention provides a composition for treating cancer comprising the monoclonal antibody, or the chimeric antibody, humanized antibody, or human monoclonal antibody, which is derived from the monoclonal antibody.

The composition for treating cancer according to the present invention may comprise an acceptable carrier according to administration modes. Preparations which are suitable for administration modes are known in the art. Further, the composition comprising the monoclonal antibody of the present invention, or the chimeric antibody, humanized antibody, or human monoclonal antibody, which is derived from the monoclonal antibody, can be administered in a pharmaceutically effective amount, and its typical administration level can be optimized using a standard clinical technique. The composition comprising the monoclonal antibody of the present invention, or the chimeric antibody, humanized antibody, or human monoclonal antibody, which is derived from the monoclonal antibody, can be administered in an amount of 0.1 mg/kg (body) to 100 mg/kg (body), preferably 0.5 mg/kg (body) to 50 mg/kg (body), and more preferably 1 mg/kg (body) to 10 mg/kg (body).

Hereinbelow, the present invention will be described with reference to

Examples.

However, Examples are provided only for the purpose of illustrating the present invention, and the scope of the present invention is not intended to be limited to Examples.

[Mode for Invention] <Example 1> Culture of cancer cells

All of cancer cell lines were cultured in a 37°C incubator with 5% carbon dioxide using the following media containing 10% fetal bovine serum (Gibco). SH-Jl (hepatocellular carcinoma), SCK (cholangiocarcinoma), Choi-CK (cholangiocarcinoma), ACHN (Renal cell adenocarcinoma), and WiDR (colorectal adenocarcinoma) were cultured in MEM (Gibco). AGS (gastric cancer), A375 (melanoma), and MDA-MB485 (mammary carcinoma) were cultured in DMEM (Gibco). SK-0V3 (ovary adenocarcinoma) was cultured in McCoy 5A medium (Gibco). A549 (non- small cell lung carcinoma) was cultured in Ham's F12K medium, and NCI- H522 (non-small cell lung carcinoma), DMS114 (small cell lung carcinoma), and NCI-H69 (small cell lung carcinoma) were cultured in an RPMI1640 medium. SH-Jl, SCK, and Choi-CK cell lines were obtained from Doctor Daegon Kim (Cheonbuk National University, College of Medicine), and other cancer cell lines were purchased from ATCC. A normal cell, hepatocyte was purchased from Cambrax, and HUVEC cell was purchased from Cambrax. The cells were cultured in a 37°C incubator with 5% carbon dioxide using an EGM-2 (Hyclone) medium containing 10% fetal bovine serum (Gibco). The peripheral blood lymphocytes (PBL) were isolated from human blood by Ficoll gradient separation, and collected.

<Example 2> Production of monoclonal antibody A10-A3 binding to cancer cell lines, Choi-CK and SCK

The cultured cancer cell lines, Choi-CK and SCK were detached using a cell dissociation buffer (Invitrogen), and about 5^χlO⁵ cells were resuspended in 30 μl of PBS. The Choi-CK cells were injected to a right paw pad of a Balb/c mouse, and after 3 days, the SCK cells were injected to the left paw pad. The injection was repeated six times at an interval of 3 to 4 days, and the cells were injected again at 1 day before the cell fusion. The FO myeloma cell line (ATCC, USA), which will be fused with lymphocytes, was cultured in the DMEM (Gibco) containing 10% fetal bovine serum 2 weeks ago.

The popliteal lymph nodes of the mice which had been immunized with the cancer cells, Choi-CK and SCK, were each taken out, washed with the DMEM (Gibco), chopped in a culture plate, and the cell suspension was transferred to a 15—ml tube. The FO myeloma cells were centrifuged, harvested, and then suspended in 10 ml of DMEM. The FO myeloma cells and the lymphocytes were counted. Thereafter, 10⁶ myeloma cells (FO) and 10⁷ lymphocytes cells were transferred to a 50- ml tube, mixed with each other, and centrifuged at 200 x g for 5 min to remove the supernatant. Then, the tube was left in a beaker filled with water at 37^°C for 2 rain. The tube was lightly tapped to soften the cell pellet, and 1 ml of a PEG solution (GIBCO) was added for 1 rain while slowly shaking the tube in water at 37°C . The tube was centrifuged at 100 xg for 2 min, and 5 ml of DMEM was slowly added thereto over 3 min. Then, 5 ml of DMEM was slowly added thereto for 2 rain, and centrifuged at 200 x g to recover the cells. In order to increase the cell fusion efficiency and viability, a hybridoma cloning factor (BioVeris, USA) was premixed with normal medium (DMEM + 20% fetal bovine serum) to be 10% final concentration. The recovered cells were gently suspended in 30 ml of the normal medium (DMEM + 20% fetal bovine serum) mixed with the hybridoma cloning factor. The cells were left in a 37°C incubator with CO2 for 30 min, and aliquotted to a 96-well plate at 10⁵ cells/70 μl per well and cultured in the 37°C incubator with CO2. At the next day, 70 μl of HAT was added to the cells, and the colonies were observed at an interval of 3 days while grown for 2 weeks or longer in the HAT media. The supernatant of the hybridoma colonies obtained by fusing the lymphocytes, which are isolated from the lymph nodes immunized with the Choi-CK cells and from the lymph nodes immunized with the SCK cells, with the myeloma cells was used for the next experiment. The clones expressing the antibody were screened using a sandwich ELISA (Enzyme Linked Immunosorbent Assay) method. 100 μl of a hybridoma culture medium was added to a plate which had been coated with an anti-mouse IgG or IgM antibody in a concentration of 2 μg/ral, and the plate was left at 37°C for 1 hour for the reaction. Then, the plate was further reacted with a 1/5,000 diluted solution of the anti- mouse IgG or IgM-HRP (horseradish peroxidase, Sigma) conjugate for 1 hour. The culture vessel was washed with a phosphate buffer added with 0.05% Tween 20, a substrate solution containing OPD (Sigma) and hydrogen peroxide (H2O2) was added thereto, and an absorbance was measured with an absorption spectrometer at a wavelength of 492 nra to screen the clones producing the antibody.

Among the above-prepared clones, the binding capacities of the hybridoma supernatants that stably secrete the antibodies were examined to the SCK and Choi-CK cells. Specifically, the cultured Choi-CK cells were treated with a cell separation buffer (GIBCO) at 37^°C for 20 min, so as to separate a single cell, and then the single cell was passed through a 40-μra strainer, and 5 x 10⁵ cells were used for flow cytometry. First, the single cells of the SCK and Choi-CK cells were suspended in PBA (PBS with 1% BSA), and the antibody supernatant was reacted at 4°C for 30 min. The cells were centrifuged at 1200 rpm at 4^°C for 5 min to remove 100 μl of the supernatant, and the cells were reacted with a 200-fold diluted anti-mouse Ig-FITC (BD) at 4°C for 30 min. Then, the reacted cells were washed with PBA twice, and propidium iodide (PΙ)-negative cells were selected. Their binding capacities to the SCK, Choi-CK cells were analyzed using a flow cytometer (FACS caliber).

Consequently, various hybridomas that secrete the antibodies binding to SCK and Choi-CK were screened, and continuously subcultured to maintain their stability for subcloning. The hybridomas secreting an A10-A3 antibody (IgGl), which have the specificity to the SCK and Choi-CK cells and maintain their stability, were screened. The hybridoma secreting the monoclonal antibody A10-A3 was designated as a hybridoma A10-A3 (Deposit No.: KCTC10909BP) , and deposited in KCTC (Korean Collection for Type Cultures, Korea Institute of Bioscience and Biotechnology, 52, Ueun-dong, Yusung-gu, Daejeon-si, Korea) on Feb. 20, 2006.

<Example 3> Analysis of binding specificity of A10-A3 antibody for various cells The A10-A3 hybridoma cell line was cultured in a serum-free medium (PFHM, Invitrogen), and antibodies were purified from the medium using a protein G-sepharose column (Pharmacia, Sweden) (Fike et al., Focus 12: 79, 1990). The binding capacity of the purified A10-A3 antibody for various cancer cells was examined in the same method as in Example 2 using fluorescence cell staining (Fig. 1). In Fig. 1, the solid line represents the monoclonal antibody A10-A3, and the filled background represents a secondary antibody only. The binding capacity of A10-A3 for various cancer cells was measured using a flow cytometer, and as a result, it was found that the monoclonal antibody specifically binds to SH-Jl, SCK, Choi-CK, AGS, NCI-H522, A549, DMSl14, NCI-H69, SK-0V3, A375 and MDA-MB485 of the cancer cells (Fig. 1). However, it was found that the monoclonal antibody does not bind to ACHN and WiDR among the cancer cells, hepatocytes, HUVECs, and peripheral blood lymphocytes (PBLs).

<Example 4> Isolation and identification of antigen that monoclonal antibody A10-A3 specifically recognizes

<Examρle 4-l> Isolation of antigen

In order to isolate a cell surface recognition molecule that the monoclonal antibody A10-A3 recognizes, the pre-cultured Choi-CK cells were washed with a PBS buffer, and biotinylated with EZ-Link SuIfo- NHS-LC-Biotin (Pierce, Rockford, IL). The cells were reacted with a lysis solution (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 μg/ml aprotinin, 100 μg/ml phenylmethylsulfonyl fluoride, and 5 μg/ml leupeptin) at 4°C for 20 min, and centrifuged to remove the cell debris. The supernatant only was recovered, and a protein concentration was determined with a BCA (bicinchoninic acid) protein detection kit (Pierce). The protein which non-specifically binds to a protein G plus sepharose (Santa Cruz Biotechnology; Santa Cruz) was reacted with 20 μl of the protein G plus sepharose in a cell lysis solution at 4°C for 2 hours, and centrifuged to recover its supernatant. The recovered supernatant was subsequently reacted with about 1 μg of the A10-A3 antibody at 4°C for 12 hours. 20 μ 1 of the protein G plus sepharose was added thereto, reacted with each other at 4°C for 2 hours, and centrifuged to recover a precipitate. The recovered precipitate was washed with the cell lysis solution ten times or more, and the remaining protein was separated in 10% SDS-PAGE. This protein was transferred to a nitrocellulose membrane, and then subjected to Western blotting. The nitrocellulose membrane was reacted in a PBST (PBS + 0.1% Tween 20) buffer containing 5% skim milk for 1 hour, and then washed with the PBST buffer twice or more. The nitrocellulose membrane was reacted with a Streptavidin-HRP (horseradish peroxidase) conjugate (1:1,500 Amersham biosciences) for 1 hour. The nitrocellulose membrane was washed with the PBST buffer five times, and the biotinylated protein was color-developed with an ECL detection reagent (Amersham biosciences). As a result, the A10-A3 antibody was found to bind to a protein having a size of about 200 kDa (Fig. 2A). In order to collect the protein imraunoprecipitated by the A10-A3 antibody, the cell lysis solution from the 1 x 10⁸ Choi-CK cells was imraunoprecipitated by the same method as described above, and the protein was separated using SDS-PAGE. This gel was stained with Coomassie G250 (Biorad).

<Example 4-2> Identification of antigen by mass spectrometry

The SDS gel containing the protein which had been immunoprecipitated by the A10-A3 antibody was stained with Coomassie G250 (BIO-RAD) according to the protocols of the Supplier. A band containing the protein was cut out of the SDS gel, washed with 30% methanol for 5 min, and chopped into small pieces. The gel pieces were reacted with 30% methanol to destain completely, and moisture was removed from the gel pieces with 100% acetonitrile for 10 min to dry in a vacuum centrifuge for 30 min. The gel piece was reacted with a 50 ii ammonium bicarbonate solution containing 300 ng of trypsin (Promega) at 37°C for 16 hours, so as to prepare the protein fragment. The peptide fragment was extracted with 100 μl of 50 mM ammonium bicarbonate three times, and dried in the vacuum centrifuge. The peptide mixture was analyzed in Q-TOF micro (MicroMass) by electrospray quadrupole time of flight tandem mass spectrometry (ESI Q-TOF MS/MS). Consequently, this protein was found to be a Ll Cell Adhesion molecule (LlCAM) (Fig. 3). In Fig. 3, the underlined parts represent the amino acid sequences which are actually identified in Q-TOF. Thus, a monoclonal antibody UJ127.11, which is the well known antibody against LlCAM, was purchased from Chemicon (USA) and immunoprecipitated using a Choi-CK cell lysis solution labeled with biotin as in Example 4-1, and identified using ECL. As shown in Fig. 2A, it can be seen that the A10-A3 antibody and the anti-LICAM antibody immunoprecipitate the protein present in the position corresponding to about 200 kDa.

<Example 4-3> Identification of LlCAM antigen by Western blotting

In order to reconfirm whether the A10-A3 antibody specifically recognizes LlCAM, the Choi-CK cell lysis solution was first immunoprecipitated with the antibody. The protein which non- specifically binds to the protein G plus sepharose (Santa Cruz Biotechnology; Santa Cruz) was prepared by reacting the cell lysis solution with 20 μ 1 of the protein G plus sepharose at 4°C for 2 hours, centrifuging the solution, and then recovering the supernatant only (preclearing in Fig. 2B), and the recovered supernatant was subsequently reacted with about 1 μg of the A10-A3 antibody at 4°C for 12 hours. 20 μ 1 of the protein G plus sepharose was added thereto, reacted with each other at 4^°C for 2 hours, and centrifuged to recover a precipitate. The recovered precipitate was washed with a cell lysis solution ten times or more, and the remaining protein was separated in 10% SDS-PAGE without 2-mercaptoethanol . This protein was transferred to a nitrocellulose membrane, and then subjected to Western blotting. The nitrocellulose membrane was reacted in the PBST (PBS + 0.1% Tween 20) buffer containing 5% skim milk for 1 hour, and then washed with the PBST buffer twice or more. The reacted nitrocellulose membrane was reacted with a well known anti-LICAM antibody UJ127 (Chemicon) as a primary antibody for 1 hour. The nitrocellulose membrane was washed with the PBST buffer five times, and then reacted with the anti-mouse IgG-horseradish peroxidase (HRP) conjugate (1:5000 Sigma) for 1 hour. The nitrocellulose membrane was again washed with the PBST buffer five times, and then color-developed with the ECL detection reagent (Amersham biosciences). As a result, it was confirmed that the anti-LICAM antibody binds to the protein having the size of about 200 kDa immunoprecipitated by the A10-A3 antibody (Fig. 2B), which indicates that the A10-A3 antibody recognizes LlCAM.

<Example 5> Epitope mapping of A10-A3 antibody

<Example 5-l> Expression of Ll immunoglobulin domain (Ig)-Fc fusion protein and Ll fibronectin type III domain (Fn)-Fc fusion protein

In order to prepare an expression vector expressing an Ll immunoglobulin domain (Ig)-Fc, a DNA sequence of a pJK^~dhfr2-Ll- monomer (as disclosed in Korean Patent Application No. 10-2006- 0079969) was used as a template, and the primers corresponding to the both ends of Ll Ig, that is, Ll-Igdom-F (5'-GAG GAG GAA TTC CGG CGC CGG GAA AGA TGG TCG TGG CG-3¹), and Ll-Igdom-R (5'-CTC CCC CTC GAG CGG CCC AGG GCT CCC CAC CAC CAA GAG CTG-3') were used to perform pretreatraent at 95°C for 5 rain. Then, DNA polymerase chain reaction (PCR) was for 30 cycles consisting of 45 sec /95°C , 45 sec/58°C , and 2 rain /72^°C and post-treatment was performed at 72°C for 10 min to amplify the DNA. A pfu polymerase (Solgent Co.) was used as the polymerase to avoid errors of the base sequence. In order to insert the amplified Ll immunoglobulin domain DNA fragment into the pJK- dhfr2-FC expression vector (Aprogen), the vector and the amplified DNA fragment were digested by EcoRI and Xhol enzymes, respectively. Electrophoresis was performed with in 1% agarose gel, and then the corresponding fragment was cut out to recover using a Gel purification kit (Intron Co.). The two recovered DNA fragments were reacted using a T4 DNA ligase (Roche) at 16°C for 12 hours, and E. coli DH5a was transformed with them using a heat shock method. An Ll immunoglobulin domain expression vector, pJK-dhfr2-LHg-FC, was prepared with the DNA isolated from the transformed cell.

In order to prepare an expression vector expressing the Ll fibronectin type III domain (Fn)-Fc, the DNA sequence of the pJK-dhfr2-Ll-monomer (as disclosed in Korean Patent Application No. 10-2006-0079969) was used as a template, and the primers corresponding to the both ends of Ll signal peptide region, that is, Fn-leader-F (5'-GA GGA GGA ATT CCG GCG CCG GGA AAG ATG GTC GTG GCG-3'), and Fn-Leader rcm-R (5'-CAC CGG CCC AGG GCT CCC CAT CAC ATG GTG TCC TTC-3') were used, and PCR was performed under the same condition as described above. The obtained Ll signal sequence fragment was recovered from the agarose gel. Further, PCR was performed with the same DNA sequence of the pJK- dhfr2-Ll-monomer as a template, and the primers corresponding to the both ends of Ll Fn, that is, Fn-dom rcm-F (5'-GAA GGA CAC CAT GTG ATG GGG AGC CCT GGG CCG GTG CCA-3'), and Fn-dom-R (5'-CTC CCC CTC GAG GAG CCT CAC GCG GCC TGT GCC ATT GGT CTT-3'), and then a Ll Fn fragment was recovered.

The recovered Ll signal sequence fragment and the Ll Fn fragment were mixed with each other in equivalent amounts, and PCR was performed with Fn-leader-F and Fn-dom-R again. The amplified Ll signal sequence-Fn DNA fragment was digested with EcoR I and Xho I enzymes, and cloned in the EcoRI-XhoI site of a JK-dhfr2-Fc expression vector (Aprogen) to prepare pJK-dhfr2-LlFn-Fc.

In order to express the Ll Ig-FC and Ll Fn-Fc fusion protein, DNAs of pJK-dhfr2-Ll Ig-Fc or pJK-dhfr2-LlFn-Fc were expressed in HEK293T (ATCC NO., CRL11268, hereinafter referred to as 293T). For this, Lipofectaraine 2000 (Invitrogen) and 20 μg of each expression vector were added to 500 μl of an Opti-MEM (Gibco BRL), respectively and reacted at room temperature for 5 min. Then, two solutions were combined, and reacted again at room temperature for 15 min. 4 ml of the Opti-MEM was added to the solution obtained by reacting Lipofectamine 2000 and DNA, and mixed. The solution was carefully added to a culture flask containing 293T cells, and cultured in the 37°C incubator with 5% carbon dioxide. After culturing for 6 hours, a medium containing 10% FBS was added to 5 ml of DMEM again, and cultured for 3 days.

<Example 5-2> Western blotting

The medium in which the 293T cells expressing Ll Ig-Fc were cultured and the medium in which the 293T cells expressing Ll Fn-Fc were cultured were separated in 7.5% SDS-PAGE, respectively, and each protein was isolated. The proteins were transferred to the nitrocellulose membrane, and Western blotting was performed. The AlO- A3 antibody, or the known anti-LICAM antibody, UJ127 (Chemicon) or 5G3 (Pharmingen) were added to the nitrocellulose membrane to react for 1 hour. Then, the nitrocellulose membrane was washed with a TBST buffer solution (TBS + 0.05% Tween 20) three times. The anti-mouse IgG- horseradish peroxidase (HRP) conjugate (1:5000 Sigma) was added to the nitrocellulose membrane to react for 1 hour. The membrane was washed with the TBST buffer solution five times, and color-developed with an ECL detection reagent (Amersham biosciences). Consequently, it was found that the A10-A3 antibody and the well known 5G3 bind to Ll Ig-Fc having the size of about 140 kDa, and the well known antibody, UJ127 binds to Ll Fn-Fc having the size of about 14OkDa (Fig. 4A).

<Example 5-3> Preparation of mutants of Ll Ig domain, and confirmation of epitope of A10-A3 antibody

In order to prepare each of the expression vectors expressing domains 1 to 5 (1-5), domains 1 to 4 (1-4), domains 1 to 3 (1-3), domains 1 and 2 (1-2), or domain 1 only, among six Ll Ig domains, PCR was performed with the pJK-dhfr2-LHg-FC clone as a template, and Ll- Igdom-F (5'-GAG GAG GAA TTC CGG CGC CGG GAA AGA TGG TCG TGG CG-3') and Ll-Ig5dom-R (5'-CTC CCC CTC GAG TTT CTT CTC GAT TGT GCT GCG-3'), Ll- Ig4dom-R (5'-CTC CCC CTC GAG GAC AAC GTA GAT GTA GGC ATT-3¹), Ll- Ig3dom-R (5'-CTC CCC CTC GAG CTC CAC GGT GAC ATA GTA CGC-3¹), Ll- Ig2dom-R (5'-CTC CCC CTC GAG CTT GAC CCG GAG GTC AAT GGG-3¹), or Ll- Igldom-R (5'-CTC CCC CTC GAG CTC GGC CAT GAG CCG GAT CTC-3') as primers, respectively.

The amplified DNA fragments were digested with EcoR I and Xho I enzymes, and inserted to the EcoRI-Xho site of the JK~dhfr2-FC expression vector (Aprogen) , respectively. Finally, pJK-dhfr2-LHgl- 5dom-Fc, pJK-dhfr2-LHgl-4dom-Fc, pJK-dhfr2-LHgl-3dom-Fc, pJK-dhfr2- LlIgl-2dom-Fc, and pJK-dhfr2-LHgldom-Fc were prepared. Further, in order to prepare an expression vector only expressing domains 2 to 6 (2-6) among six Ll Ig domains, PCR was performed with the pJK-dhfr2- LlIg-Fc clone as a template, and the Ll-Igdom-F and the Lldellgl-spR (5'-TGG CCA CTT GGG GGC ACC GAT CTG GATBAAG CAG GCA-3¹). Further, PCR was performed with Ll-Ig2-6dom-F (5'-TGC CTG CTT ATC CAG ATC GGT GCC CCC AAG TGG CCA-3¹) and the Ll-Igdom-R as primers. Then, PCR was again performed with the amplified DNA fragments using Ll-Igdom-F and Ll-Igdom-R, and the obtained DNA fragments were digested with EcoR I and Xho I enzymes, respectively. The digested fragments were cloned in the EcoRI-XhoI site of a JK-dhfr2-Fc expression vector (Aprogen) to prepare pJK-dhfr2-LlIg2-6dom-Fc.

In order to obtain a fusion protein that the Ig 1-5, 1-4, 1-3, 1-2, 1, and 2-6 domains of Ll are fused with Fc, the expression vectors were expressed in 293T cells, respectively, and then each of the culture media was subject to Western blotting using the A10-A3 antibody, or the 5G3 antibody. As a result, it was found that the A10-A3 antibody binds to all of the domains 1-2, 1-3, 1-4, and 1-5 including the domain 1 among each of Ll-Fc fusion proteins (Fig. 4B). However, the A10-A3 antibody did not bind to Ll Ig 2-6 excluding the domain 1. These results indicate that the A10-A3 antibody binds to the domain 1 of Ll. On the other hand, it was found that the 5G3 antibody binds to the domains 1-5, 1-4, 1-3, and 1-2 except for the domain 1. These results_indicate that the 5G3 antibody binds to the domain 2 of Ll (Fig. 4C). Conclusively, these results indicate that the A10-A3 antibody is different from UJ127 and 5G3.

<Example 6> Immunohistochemical staining of A10-A3 antibody to cancer tissues For immunohistochemical staining, tissue fragments were cleaved from each cancer in a thickness of 3μm, and were adhered to slides coated with poly-L-lysine. First, the slides were dried in an oven at 60°C for 3 hours, and deparaffinated in xylene three times at room temperature for 5 min. The slides was treated with 100%, 90%, 80%, and 70% alcohol for 1 min, respectively, and dipped in a target retrieval solution (DAKO, Carpinteria, CA) to restore antigenicity. Then, the slides were washed with a TBST (Tris-buffered saline-Tween 20) buffer, which had been boiled in a pressure cooker for 4 min. For high-sensitivity of immunohistochemical staining, a Biotin-free Tyramide Signal Amplification System, CSA II kit (DAKO, Carpinteria, CA), was used. In order to remove the non-specific antigen, it was reacted with 3% hydrogen peroxide for 5 min, washed with a buffer for 5 min twice, and in order to remove non-specific protein bindings, it was washed with a sufficient serum-free protein block for 5 min. As a primary antibody, the A10-A3 antibody (1:50 dilution) was applied, reacted for 15 min, and treated with anti-mouse immunoglobulin-HRP for 15 min. Thereafter, it was left in an amplification reagent for 15 min, and then treated with anti-fluorescein-HRP for 15 min. DAB(diaminobenzidine) was used to develop the color for 5 min, and then counter staining was performed using Meyeril hematoxylin. After each step ends, the resultant was washed with the TBST buffer twice for 5 min. The same procedure was performed for a negative control, except that upon staining, a normal sheep serum was added without the primary antibody, or a normal mouse IgGl serum was added instead of the primary antibody. As a result, it was found that the antibody does not bind to a normal tissue, but it readily binds to the tissues of cholangiocarcinoma, lung cancer, breast carcinoma, ovarian carcinoma, skin cancer, colon cancer, and pancreatic cancer (Fig. 5). These results indicate that LlCAM is expressed in the tissues of cholangiocarcinoma, lung cancer, breast carcinoma, ovarian carcinoma, skin cancer, colon cancer, and pancreatic cancer. Therefore, in the present invention, the effect of the A10-A3 antibody on the proliferation or metastasis of various cancer cells was analyzed.

<Example 7> Inhibition of cancer cell growth by A10-A3 antibody

In order to determine whether the antibody against LlCAM inhibits the cancer cell growth, Choi-CK, SCK, DMSl14, and SK-0V3 cells, which the A10-A3 antibody binds to, and ACHN cells as a negative control were cultured in each well of a 6-well plate at 2 x 10⁵ cells/well in 3 ml of a medium, and the monoclonal antibody was added to each well at a concentration of 10 μg/ml . The cells were reacted in a CO2 reactor at 37°C for 72 hours. Then, the cells were recovered, and the living cells and the dead cells were each counted in a 0.2% Tryphan Blue solution, to determine a percentage of the living cells in the total cells. As a result, it was found that the A10-A3 antibody reduced the growth of Choi-CK, SCK, DMSl14, and SK-0V3 cells, and did not any influence on the ACHN cells (Fig. 6A). These results indicate that the A10-A3 antibody suppresses the growth of cholangiocarcinoma, lung cancer, and ovarian carcinoma. When the well-known antibodies, UJ127 (Chemicon) and 5G3 (Pharmingen) , which are known to specifically bind to LlCAM and known to suppress the growth of the cell lines of breast carcinoma, colon cancer, and cervical cancer, were treated to cholangiocarcinoma cells and small cell lung cancer cells, it was observed that the UJ127 antibody suppressed the growth of the cancer cells. On the other hand, the 5G3 antibody slightly suppressed the growth of the cholangiocarcinoma cells (Choi-CK), but did not suppress the growth of lung cancer cells (DMS114) at all (Fig. 6B). The 5G3 antibody bound to the Choi-CK and DMS114 cells (Fig. 1C). These results demonstrated that in some cases, the monoclonal antibody binding to LlCAM may not inhibit the proliferation of cancer cells.

<Example 8> Inhibition of invasion and migration of cancer cells by AlO- A3 antibody In order to perform invasion assay, a QCM 24-well cell invasion assay kit (CHEMICON) was used. To rehydrate the ECM layer of an insert, 300 μl of a pre-warmed serum free medium (RPMI, 10 mM HEPES, pH 7.4) was put into the insert, and left to stand at room temperature for 30 min. The Choi-CK, SCK, SK-0V3, and ACHN were washed with PBS twice, and 3 ml of trypsin-EDTA was added thereto to put into the 37°C incubator. The cells in the invasion medium (RPMI, 10 mM HEPES pH 7.4, 0.5% BSA) were taken out, and the number of the cells was adjusted to 1 x 10⁵ cells/200 μl with the invasion medium. Then, the cells were put into each insert, and treated with the A10-A3 antibody, or the known antibody 5G3 (10 μg/ml), and normal mouse IgG (10 μg/ml). To a lower chamber, an invasion medium containing 10% FBS was put, and cultured at the 37°C incubator for 72 hours. After culturing, the cells remaining in the inserts and the media were removed, and the inserts were transferred to fresh wells. The inserts were put onto 225 μl of a pre-warmed cell lysis solution, and cultured at the 37°C incubator for 30 min. The inserts were shaken to completely separate the remaining cells, and 75 μl of the Lysis buffer/Dye solution were put into the solution containing the cell lysis solution and the cells, and left at room temperature for 15 min. 200 μl of the solution was transferred to a 96-well, and the fluorescence was read at 480 nm/520 nm. As a result, it was found that the A10-A3 antibody does not suppress the ACHN, but suppresses the invasion of the cancer cells in the Choi-CK, SCK and SK-0V3 cells (Fig. 7A). However, the known antibody, 5G3, did not suppress the invasion of DMS114 (Fig. 7A). In migration assay, the same procedure was carried out for experiment, except that the bottom of the insert was coated with 10 μg/ml of collagen type I. As a result, it was found that the A10-A3 antibody does not have an inhibitory effect on the ACHN, but inhibits the migration of the Choi-CK, SCK, and SK-0V3 cells (Fig. 7B).

<Example 9> Induction of cancer cell apoptosis by A10-A3 antibody In order to determine whether the A10-A3 antibody induces cancer cell apoptosis, immunocytochemical analysis was performed on two cancer cells, Choi-CK and SCK, using Annexin V-FITC (BD Pharmingen). If the apoptosis is induced, phosphatidylserine comes from the internal membrane of the cell to the outer membrane, and allows Annexin V to bind to the dead cells. 10,000 cancer cells were put into each slide chamber, cultured for 9 hours, and the A10-A3 (10 μg/ml) antibody and the mouse IgG (10 μg/ml) were used to treat the cells. The cells which had been cultured overnight in the slide chamber were washed with a Ca²⁺-Mg²⁺-PBS buffer, and fixed with 4% paraformaldehyde. The slide was washed with the buffer six times. The cells were treated with a blocking solution (10% normal horse serum and 0.1% bovine serum albumin in PBS) at room temperature for 1 hour, and Annexin V-FITC (BD Biosciences) was diluted in the ratio of 1:1000 with an Annexin V-FITC binding solution [0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 niM CaCl₂], and bound to the cells for 40 min. The slide was washed with a buffer twice, mounted in Vectashield (Vector), and then analyzed with a fluorescence microscope. As a result, it was found that the Annexin V clearly binds to the cells treated with the A10-A3 antibody, but does not bind to the control treated with the mouse immunoglobulin IgG antibody (Fig. 8A).

Alternatively, in order to measure the apoptosis, the expression of the activated casρase-3, as a measurement of apoptosis, was determined on the Choi-CK, SCK, and ACHN cells. For this, a FITC-conjugated monoclonal active caspase-3 antibody apoptosis kit (BD Bioscience) was used. To the cultured Choi-CK, SCK, and ACHN cells, the A10-A3 antibody and the normal mouse IgG antibody were added in a concentration of 10 μg/ml. Then, the cells were reacted in a CO₂ reactor at 37°C for 48 hours. The cells were treated with a cell separation buffer (GIBCO) at 37°C for 20 min, and a single cell was separated therefrom. Then, the single cells were passed through a 40- μm strainer, and 1 x 10⁵ cells were used for flow cytometry. First, the single cells of the SCK, Choi-CK, and ACHN cells were suspended in Cytofix/Cytoperm, and reacted for 20 min in ice. The cells were precipitated, and then the supernatant was taken out, and the precipitates were washed with a Perm/Wash solution three times. 20 μl of the caspase-3 antibody was added to each of the control and the experimental group of the cells, and reacted at room temperature for 30 min. The cells were precipitated, and the supernatant was taken out and washed with a Perm/Wash solution twice. The cells prepared were subject to flow cytometry. As a result, it was found that the casρase-3 antibodies bind to the SCK and Choi-CK cells treated with the A10-A3 antibody, but the caspase-3 antibody does not bind to the ACHN cells to which the A10-A3 antibody does not bind (Fig. 8B).

The above two results indicate that the A10-A3 antibody induces the cancer apoptosis.

<Example IO Inhibition of signal transduction of cancer cells by A10-A3 antibody

In order to determine whether the inhibitory effects of the A10-A3 antibody on the proliferation, migration, and invasion of cholangiocarcinoma cells are caused by the inhibition of signal transduction of the cancer cells, the quantitative change in cell signaling molecules, which play important roles in proliferation, migration, invasion and survival of the cancer cells (e.g., PCNA, phosphorylated MAPK, AKT, and FAK), was analyzed after treating the antibody.

<Example 10-l> Inhibition of PCNA expression in cancer cells by A10-A3 antibody

In order to confirm whether the expression of a PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA), which indicates cell proliferation, is reduced by the A10-A3 antibody, the Western blotting was performed. The Choi- CK cells were treated with 10 μg of each of the A10-A3 antibody and the mouse IgG for 72 hours, and the cells were collected and dissolved in the cell lysis solution. A BCA (bicinchoninic acid) protein detection kit (Pierce) was used to determine the concentration of the protein, and 40 μg of the protein was run on 8% SDS-PAGE. The protein was transferred to a nitrocellulose membrane at 25 V for 90 min. The membrane was blocked with 5% skim milk overnight at 4°C , and reacted with a mouse monoclonal anti-PCNA antibody (Novocastra Laboratories 1:500), and an anti-β actin antibody (Oncogene, 1:4000) for 1 hour. Then, it was reacted with the anti-mouse horseradish peroxidase- conjugated antibody (Cell Signaling, 1:1000), and washed with PBST, and PCNA and β-actin were detected using an enhanced chemi luminescence reagent (ECL) (Amersham Pharmacia Biotech). It was observed that only in the Choi-CK cells treated with the A10-A3 antibody, the expression of PCNA is significantly reduced (Fig. 9A).

<Example 10-2> Inhibition of MAPK phosphorylation by A10-A3 antibody In order to confirm whether MAPK (mitogen-activated protein kinases) phosphorylation, which is associated with the growth, invasion, and survival of the cancer cells, is reduced by the A10-A3 antibody, the Western blotting was performed. The Choi-CK cells were treated with 10 μg/ml of each of the A10-A3 antibody and the mouse IgG for 72 hours, and then collected and dissolved in the cell lysis solution. The BCA (bicinchoninic acid) protein detection kit (Pierce) was used to determine the concentration of the protein, and 40 μg of the protein was run on 12% SDS-PAGE. The protein was transferred to a nitrocellulose membrane at 25 V for 90 min.

The membrane was blocked with 5% skim milk overnight at 4°C , and reacted with a rabbit polyclonal anti-phospho MAPK antibody (Ab Cam 1:1000) in 1% skim milk. Further, in order to examine the expression of the unphosphorylated MAPK, the nitrocellulose membrane, which is blocked with the same amount of protein by the same treatment as described above, was reacted with an anti-MAPK (Ab Cam, 1:1000) antibody for 1 hour. Further, it was reacted with an anti-rabbit horseradish peroxidase- conjugated antibody (Cell Signaling, 1:10000) for 1 hour, washed with PBST, and then the phospho MAPK and MAPK were detected using ECL (Amersham Pharmacia Biotech). In the Choi-CK cancer cells expressing the same MAPK, it was observed that the amount of the phospho-MAPK was significantly reduced only in the Choi-CK treated with the A10-A3 antibody (Fig.9A).

<Examρle 10-3> Inhibition of AKT phosphorylation by A10-A3 antibody In order to confirm whether the Akt phosphorylation, which is involved in the survival of cancer cells, is reduced by the A10-A3 antibody, the Western blotting was performed. The Choi-CK cells were treated with 10 μg of each of the A10-A3 antibody, and the mouse IgG, each for 30 min, 1 hour, 1 hour and a half, and 2 hours, and the cells were collected and dissolved in the cell lysis solution. The BCA (bicinchoninic acid) protein detection kit (Pierce) was used to determine the concentration of the protein, and 40 μg of the protein was run on 12% SDS-PAGE. The protein was transferred to a nitrocellulose membrane at 25 V for 90 min. The membrane was blocked with 5% skim milk overnight at 4°C , and reacted with a rabbit polyclonal anti-phospho Akt antibody (Ab Cam 1:1000) and a rabbit polyclonal anti-Akt antibody (Ab cam 1:1000) in 1% skim milk overnight. Further, the membrane was reacted with anti-rabbit IgG antibody-HRP

(Santa Cruz 1:10000) for 1 hour. The membrane was washed with PBST, and the phospho Akt and the total Akt were detected using the enhanced chemi luminescence reagent (ECL) (Amersham Pharmacia Biotech). It was observed that the amount of the phospho Akt is reduced in the Choi-CK treated with the A10-A3 antibody (Fig. 9B).

<Example 10-4> Inhibition of FAK phosphorylation by A10-A3 antibody

In order to confirm whether the focal adhesion kinase (FAK) phosphorylation, which essentially acts on the activation of integrin playing an important role in the growth and migration of cancer cells, is reduced by the A10-A3 antibody, the Western blotting was performed. The Choi-CK cells and SCK cells were treated with the A10-A3 antibody each for 30 min, 1 hour, 1 hour and a half, and 2 hours, and the cells were collected and dissolved in the cell lysis solution. The BCA (bicinchoninic acid) protein detection kit (Pierce) was used to determine the concentration of the proteins, and 40 mg of the proteins were run on 7.5% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane at 25 V for 90 min. The membrane was blocked with 5% skim milk overnight at 4°C , and reacted with a rabbit polyclonal anti-phospho FAK antibody (Ab cam 1:1000) in 1% skim milk overnight. Further, the membrane was reacted with an anti-β-actin antibody (Oncogene, 1:4000) for 1 hour. Then, the membrane was reacted with an anti-rabbit horseradish peroxidase- conjugated antibody (Cell Signaling, 1: 10000) for 1 hour, washed with PBST, and the phospho FAK and β-actin were detected using the enhanced chemi luminescence reagent (ECL) (Amersham Pharmacia Biotech). It was observed that the amount of the phospho FAK is reduced in the Choi-CK and SCK treated with the A10-A3 antibody (Fig. 9C). These results indicate that the inhibitory effect of the A10-A3 antibody on the growth and metastasis of cancer cells is associated with the inhibition of the integrin activity.

In fact, the integrin expression in the Choi-CK, and SCK cells were analyzed, and as a result, it was found that αvβ5 is expressed (Fig.

10A), the cell growth is inhibited by treating the cells with the monoclonal antibody against αvβ5 (Ab Cam), and the degree of inhibition by it is similar to by the A10-A3 antibodies (Fig. 10B).

These results indicate that LlCAM is involved in the growth and metastasis of the cancer cells via the integrin αvβδ.

<Example 11> Confirmation of inhibitory effect of A10-A3 antibody on cancer cells proliferation induced by soluble LlCAM LlCAM is integrated in the cell membrane, but in some cases, it is cleaved by a proteolytic enzyme such as ADAMlO and plasmin to be secreted out of the cell. The secreted soluble LlCAM binds to integrin of the cell to promote the migration and survival of cancer cells (Biochem. Biophys. Res. Commun. 1997; 232: 236-239). Thus, it was analyzed to determine whether the A10-A3 antibody of the present invention binds to the secreted LlCAM and inhibits the growth of cancer cells induced by the secreted LlCAM.

<Example 11-1> Expression and purification of soluble LlCAM-Fc In order to express the soluble LlCAM, pJK-dhfr2-Ll-FC DNA was expressed in HEK293T, as in Example (5-1). After culturing for 48 hours, the culture medium was collected, and spinned using a protein G-sepharose column (Pharmacia, Sweden) at 4°C for 2 hours for the antibody's binding, the column was allowed to stand vertically, and the wall of the column was washed with a washing liquid (0.5 M NaCl, 0.1 M Tris, pH 8.0) using a serum separator tube. The column was connected to a peristatic pump, and sufficiently washed with a washing liquid. After completion of washing, the antibodies were eluted with 0.2 M glycin-HCl (pH 2.7). At this time, the eluting solution was buffered in the previously prepared tube containing 1 M Tris (pH 9.0). Then, dialysis was repeatedly performed 4 times in PBS (pH 7.4).

<Example ll-2> Inhibitory effect of A10-A3 antibody on cancer cells proliferation by soluble LlCAM

In order to confirm that the soluble LlCAM plays a role in the proliferation of the cholangiocarcinoma cells (Choi-CK, SCK), and that treatment with the A10-A3 antibody suppresses the proliferation of cancer cells by inhibiting action of the soluble LlCAM, 3,000 Choi-CK cells, and 2,000 SCK cells per well were plated in a 96-well plate. Further, the water-soluble Ll-Fc having a concentration of 1 μg/ml , 5 μg/ml, 10 μg/ml, or 20 μg/ml was mixed with the A10-A3 antibody having the same concentrations, and left to stand at room temperature for 5 min. The mixed solution was added to the previously prepared Choi-CK cells and the SCK cells. The cells were cultured for 50 hours, and WST-I was added thereto, and then their absorbance was measured at 450 nm. As a result, in the cells treated with the water-soluble LlCAM, their growth was found to increase. In the Choi-CK cells and the SCK cells treated with the mixture of the A10-A3 antibody and the water- soluble LlCAM, their growth was found to be inhibited (Fig. 11).

<Example 12> Experiment on inhibition of cancer cells growth by A10-A3 antibody in mouse model A nude mouse Balb/c nu/nu was purchased from Japan SLC, Inc through Central Lab. Animal Inc. It was 6 to 8-week old, and had a body weight of 18 to 22 g, and domesticated in Korea Institute of Bioscience and Biotechnology for 1 week. Thereafter, 3 x 10⁶ Choi-CK cells were implanted subcutaneousIy in the mouse, and at day 20, they were grown to a size of 390 mm³ (Fig. 12A). A tumor volume was measured in a dimension of width (mm) x length (mm) x height (mm)/2. At a final day for experiment, the nude mouse was sacrificed with CO2, and then the tumor was separated therefrom, and weighed. In order to examine its toxicity to the animals, the body weight of the animal was measured. Standard variances (SDs) and p values were calculated using ANOVA (Prisim, GraphPad software, USA) and a student t test. When A10-A3 had been injected to the tail vein of the animal from the day 1, three times per week at a concentration of 10 mg/kg, a strong anti-cancer effect was observed until the day 20 (Fig. 12A). For the control, the same amount of mouse IgG antibody was injected to the tail vein of the animal. At the day 20, the tumor size was 232 mm³. That is, 40% of the anti-cancer effect was observed as compared to the control (Fig. 12A). At the final day for experiment (day 20), the tumor was separated and weighed (Fig. 12B). The average tumor weight of the control was 872 mg, and the average tumor weight of the group administered with the A10-A3 antibody was 516 mg. Consequently, 40% of the anti-cancer effect was observed.

In order to examine the toxicity of the A10-A3 antibody, the change in the body weight of the nude mouse was measured for 20 days, and the behavior change was also examined with naked eyes (Fig. 12C). At the day 20, as compared with the control, the reduction in the body weight and abnormal behavior were not observed.

[Industrial Applicability] As shown from the above, the monoclonal antibody of the present invention binds to LlCAM protein which is present on the surface of various cancers such as cholangiocarcinoma and lung cancer, or secreted therefrom, and thus inhibits the growth, invasion, or migration of the cancer cells, and induces apoptosis of cancer cells. Therefore, it can be useful for treatment and diagnosis of cancers.

Claims

[CLAIMS]

[Claim 1]

A monoclonal antibody which selectively binds to the first immunoglobulin-like domain (IgI) of LlCAM to inhibit the proliferation, migration, or invasion of a cancer cell, or a fragment containing the antigen-binding site of the antibody.

[Claim 2]

The monoclonal antibody or the fragment thereof according to claim 1, wherein the LlCAM is a membrane-bound form or a free form secreted from the cell membrane.

[Claim 3]

The monoclonal antibody or the fragment thereof according to claim 1 or 2, which is secreted from a hybridoma of the deposit No. KCTC 10909BP.

[Claim 4]

The monoclonal antibody or the fragment thereof according to claim 1 or 2, wherein the cancer cell is selected from the group consisting of a cholangiocarcinoma cell, a lung cancer cell, a breast carcinoma cell, an ovarian carcinoma cell, a skin cancer cell, a colon cancer cell, and a pancreatic cancer cell.

[Claim 5]

The monoclonal antibody or the fragment thereof according to claim 1 or 2, wherein the fragment of the antibody is selected from the group consisting of Fab, F(ab'), F(ab')2, and Fv.

[Claim 6] A hybridoma of the deposit No. KCTC 10909BP.

[Claim 7]

A composition for treating cancer comprising the monoclonal antibody or the fragment thereof according to claim 1, or a chimeric antibody, a humanized antibody, or a human monoclonal antibody produced therefrom.

[Claim 8]

The composition for treating cancer according to claim 7, wherein the cancer is selected from the group consisting of cholangiocarcinoma, lung cancer, breast carcinoma, ovarian carcinoma, skin cancer, colon cancer, and pancreatic cancer.

[Claim 9]

The composition for treating cancer according to claim 7, wherein a well-known agent for treating cancer binds additionally to the antibody.

[Claim 10]

A method for treating cancer using the monoclonal antibody or the fragment thereof according to claim 1 or a chimeric antibody, a humanized antibody, or a human monoclonal antibody produced therefrom.