WO2009045075A2

WO2009045075A2 - A monoclonal antibody specific to human embryonic stem cell, a hybridoma secreting the same and a method for detecting or isolating non-differenced embryonic stem cell

Info

Publication number: WO2009045075A2
Application number: PCT/KR2008/005832
Authority: WO
Inventors: Hyo Jeong Hong; Yeon Sung Son
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-10-02
Filing date: 2008-10-02
Publication date: 2009-04-09
Also published as: KR20090034242A; KR101302711B1; WO2009045075A3

Abstract

Disclosed herein is the use of L1CAM, which is a marker protein of undifferentiated human embryonic stem (ES) cells. Also disclosed are an antibody binding specifically to L1CAM, a hybridoma secreting the antibody, and a method of detecting, identifying or isolating undifferentiated human ES cells using the antibody. Since antibodies binding specifically to L1CAM, including a novel antibody 4-63 according to the present invention, bind specifically to the L1CAM protein expressed on undifferentiated human ES cells, they are useful in the accurate analysis of characteristics of undifferentiated human ES cells and in isolating human ES cells for cell therapy.

Description

[DESCRIPTION]

[invention Title]

A MONOCLONAL ANTIBODY SPECIFIC TO HUMAN EMBRYONIC STEM CELL, A HYBRIDOMA SECRETING THE SAME AND A METHOD FOR DETECTING OR ISOLATING NON-DIFFERENCED EMBRYONIC STEM CELL

[Technical Field]

The present invention relates to a marker protein, LlCAM, which is expressed on the surface of undifferentiated human embryonic stem cells, a monoclonal antibody binding specifically to the protein, and a hybridoma secreting the antibody. The present invention is also concerned with a method of preparing the monoclonal antibody, and a method of detecting or isolating undifferentiated human embryonic stem cells using the monoclonal antibody.

[Background Art]

Stem cells have the ability to differentiate into various cell types constitutingtissuesofalivingbody, and collectively the term means undifferentiated cells prior to being differentiated, which can be obtained from the embryo, the fetus and each tissue of the adult body. Stem cells are typically divided into pluripotent embryonic stem cells and multipotent adult stem cells. Embryonic stem cells are cells derived from the inner cell mass, which will eventually form the fetus, within the blastocyst during early embryogenesis, and have the potential to virtually differentiate into every cell type of every tissue found in the body. That is, embryonic stem cells are undifferentiated cells that are capable of proliferating indefinitely, differentiating into all cell types, and giving rise to germ cells, which are inherited by the next generation, unlike adult stem cells.

Studies on embryonic stem cell culture started in 1981 when a method of cultivating mouse embryonic stem cells was first established (Evans et al. , Nature, 292: 151-156, 1981) . In 1996, a method of cultivating pluripotent embryonic stem cells from primates was developed (Thomson et al . , Biol . Reprod. , 55 : 254-259, 1996) . In 1998, Thomson et al. established in the U.S.A. a method of cultivating human embryonic stem (ES) cells (Thomson et al., Science, 282: 1145-1147, 1998) . Since human embryonic stem cells have a special quality of pluripotency (the ability to develop into all cell types) and can be cultivated to proliferate in the presence of feeder layers or conditioned media from feeder layers, they are able to differentiate into cell types of all tissues when induced to form embryoidbodies (EBs) (Thomsonet al . , Science, 282: 1145-1147, 1998; Reubinoff, et al., Nat. Biotech. ,18:399-404; Park, et al . , Biol. Reprod. 69:2007-2014, 2003) . Human ES cells may be thus used to replace specific cells or organs damaged by diseases or accidents after being induced to differentiate into the specific cells or cells specific to the organs. With the expectation, human ES cells are receiving increasing attention as a promising therapeutic approach for various incurable diseases. Human ES cells have been identified with the expression of intracellular markers, Oct-4, Nanog and Sox-2, which are necessary to maintain self-renewal and pluripotency, and with antibodies to cell surface antigens TRA-1-60, TRA-1-81, SSEA3, SSEA4, and the like. These antibodies recognize molecules which mostly possess carbohydrate epitopes and whose functions have been known not to be essential to maintain the self-renewing and pluripotent phenotypes of human ES cells (Badcock, et al. , Cancer Res. 59:4715-4719, 1999; Kannagi et al., EMBO. J. 2:2355-2361, 1983; Brimble et al., Stem Cells 25:54-62, 2007) . Thus, there is a need to find many more cell surface markers expressed on human ES cells for studies or isolation of undifferentiated human ES cells.

For application of various specialized functional cells into which human ES cells are induced to differentiate in cell therapy, it is important to isolate the specialized cells with high purities and to virtually ensure efficacy and safety thereof in animals or humans. Human ES cells, however, have been known to form tumors inmice (Thomson et al ., Science, 282: 1145-1147, 1998; Reubinoff, et al., Nat. Biotech., 18:399-404; Park, et al., Biol. Reprod. 69:2007-2014, 2003) . Thus, specialized cells differentiated from human ES cells must be used for cell therapy after human ES cells have been completely removed. Therefore, the development of many more antibodies specifically recognizing human ES cells may give rise to accurate analysis of characteristics of human ES cells and complete removal of human ES cells in cell therapy. [Disclosure] [Technical Problem]

It is therefore an object of the present invention to provide an antibody that binds specifically to a marker protein of human ES cells, LlCAM, and a hybridoma that produces the antibody.

It is another object of the present invention to provide a method of identifying or isolating undifferentiated human ES cells using an antibody binding specifically to LlCAM.

It is a further object of the present invention to provide a method of removing undifferentiated human ES cells using an antibody binding specifically to LlCAM.

[Technical Solution]

The inventors of this application cultivated human ES cells and characterized the cultured human ES cells in the undifferentiated state . The cultured cells, identified as being in the undifferentiated state, were then used to prepare a monoclonal antibody specific to human ES cells . The monoclonal antibody thus-produced was found to bind specifically to a cell surface protein on human ES cells . As well, the present inventors found that the cell surface protein of human ES cells, to which the monoclonal antibody binds, is LlCAM, which is a marker protein that functions to maintain human ES cells in an undifferentiated state. The successful isolation of human ES cells was done with the monoclonal antibody, thereby leading to the present invention. [Advantageous Effects]

In accordance with the present invention, the monoclonal antibody of the present invention binds specifically to LlCAM, which is expressed on undifferentiated human ES cells. Thus, the monoclonal antibody is very useful in identifying and isolating undifferentiated human ES cells. As well, the monoclonal antibody is useful for the removal of undifferentiated human ES cells from their differentiated progeny in cell therapy.

[Description of Drawings] FIG. 1 shows the marker expression in human ES cells to ascertain the undifferentiated phenotype. (A) A human ES cell culture was characterized by hematoxylin & eosin staining (1), alkaline phosphatase activity (2), and immunohistochemical staining with anti-SSEAl (3), anti-SSEA3 (4) and anti-SSEA4 (5) antibodies; (B) Flow cytometric analysis with anti-SSEAl, anti-SSEA3 and anti-SSEA4 antibodies; (C) RT-PCR for expression of pluripotent transcription factors, Oct4, Nanog and Sox2.

FIG. 2 shows through immunofluorescent staining that a monoclonal antibody, 4-63, according to the present invention binds to the surface of human ES cells. Flow cytometric analysis revealed that the 4-63 antibody binds to human ES cell lines, Miz-hESl, SNUhES3 and HSF6 (A) and does not bind to mouse embryonic stem cells (Jl) and mouse embryonic fibroblasts (MEF) (solid line: staining with the 4-63 antibody; red background: staining with a secondary antibody alone; anti-SSEAl: a negative control marker not binding to human ES cells; anti-SSEA3 and anti-SSEA4 : positive control markers binding to human ES cells) . Immunofluorescent staining with the 4-63 antibody and an antibody against TRA-1-81, which is a known cell surface marker for undifferentiated human ES cells, revealed that both antibodies bind to a human ES cell line, HSF6 (C) .

FIG. 3 shows the results of immunoprecipitation (IP) for detection of a molecule binding to the 4-63 antibody. (A) A human ES cell line, Miz-hESl, was biotinylated on the surface thereof and immunoprecipitated with the 4-63 monoclonal antibody. The immunoprecipitated protein was separated on 10% SDS-PAGE and detected with Western blotting using streptavidin-HRP. (B) H9, HSF6 and SNU-hES3 cells were lysed and immunoprecipitated with the 4-63 antibody and a commercially available anti-LICAM monoclonal antibody (5G3) . The immunoprecipitated protein was separated on 10% SDS-PAGE and detected with Western blotting using the 4-63 antibody biotin-labeled and streptavidin-HRP.

FIG. 4 shows the results of Q-TOF analysis. The protein immunoprecipitated from human ES cells was excised from a 10% SDS-PAGE gel and subjected to in-gel trypsin digestion. The peptides were analyzed through ESI-Q-TOF MS/MS, and the antigen recognized by the 4-63 antibody was identified as Ll cell adhesion molecule (LlCAM) .

FIG. 5 shows the results of epitope mapping. (A) Western blotting with 4-63, 5G3 and UJ127 antibodies for an LlCAM immunoglobulin (Ig) domain- or fibronectin type III (Fn) -human Ig Fc fusion protein; (B) Western blotting with the 4-63 antibody for soluble LlCAM (SoILl) and human Ig Fc fusion proteins of deletion mutants of LlCAM Ig domains, that is, domains 1 to 6 (Igl-6) , domains 1 to 5 (Igl-5) , domains 1 to 4 (Igl-4) , domains 1 to 3 (Igl-3) , domains 1 to 2 (Ig 1-2) and domain 1 alone (IgI) ; (C) Western blotting with 5G3 antibody for the deletion mutants of LlCAM Ig domains.

FIG. 6 shows through immunofluorescent staining the decreased binding specificity of the 4-63 monoclonal antibody according to the present invention to the differentiated state of human ES cells. (A) Like anti-SSEA3 and anti-SSEA4 antibodies, the 4-63 antibody exhibited decreased binding specificity to human ES cells differentiated into embryoid bodies. (B) When human ES cells were exposed to retinoic acid to be induced into a differentiated state, the 4-63 antibody exhibited decreased binding specificity to the ES cells. Also, markers for the undifferentiated phenotype of human ES cells, SSEA3, TRA-1-60 and TRA-1-81, which was used as positive controls, were down-regulated in the presence of retinoic acid.

FIG. 7 shows the results of RT-PCR for expression patterns of pluripotent transcription factors, Nanog, Oct4 and Sox2, ectodermal (Paxβ), mesodermal (CD34) and endodermal (AFP) markers, and LlCAM in human ES cells in an undifferentiated state and ina stateofbeing induced to differentiate into embryoid bodies . FIG. 8 shows the results of FACS sorting, denoting that the 4-63 antibody enables the isolation of undifferentiated human ES cells.

[Best Mode] In one aspect, the present invention provides a monoclonal antibody that binds specifically to LlCAM, which is a cell surface protein of undifferentiated human embryonic stem (ES) cells.

As used herein, the term "monoclonal antibody" is meant to indicate a protein molecule that is directed against a single antigenic region (single epitope) and binds specifically thereto .

Monoclonal antibodies may be prepared using a fusion method, which is widely known in the art (Kohler et al. , European Journal of Immunology 6:511-519) . A hybridoma cell secreting a desired monoclonal antibody is typically prepared by fusing immune cells from an immunologically suitable host animal, such as mice, injected with a protein as an antigen, with a carcinoma cell line. Cells of the two groups are fused with each other using a method known in the art, for example, using polyethylene glycol, and antibody-producing cells are propagated using a standard culture method. After uniform cell colonies are obtained through subcloning using a limited dilution technique, a hybridoma cell capable of producing an antibody specific to the antigen is cultivated in large scale in vitro or in vivo. The monoclonal antibody of the present invention binds specifically to a protein of undifferentiated human ES cells having a molecular weight of about 220 kDa on 10% SDS-PAGE.

In detail, the monoclonal antibody of the present invention recognizes Ll cell adhesion molecule (LlCAM) (FIG. 4) . It will be understood by those skilled in the art that the monoclonal antibody of the present invention may be modified to chimeric, humanized and human monoclonal antibodies so as to reduce the potential for immunogenicity in humans. These chimeric, humanized and human monoclonal antibodies may be readily generated from the monoclonal antibody of the present invention using a known method, forexample, using a methodbased on grafting a variable region of the monoclonal antibody of the present invention, particularly a complementarity-determining region (CDR) or only a specificity-determining residue (SDR) within the CDR onto a human antibody framework. Such variants are contemplated as within the scope of the present invention. Also, the present invention includes whole antibodies having two full-length light chains and two full-length heavy chains as well as functional fragments of antibody molecules, so long as they retain the aforementioned binding properties. The functional fragments of antibody molecules are intended to mean fragments retaining at least an antigen-binding capacity, and may include Fab, F(ab'), F(ab')₂ and Fv.

In one embodiment, in order to produce a monoclonal antibody specific to undifferentiated human ES cells, the present inventors cultivated human ES cells in a large scale using collagenase to facilitate the follow-up culture, analyzed the characteristics of the human ES cells to identify the cultured cells as being human ES cells, and immunoinjected the cultured human ES cells into mice.

In detail, human ES cells were cultured, subjected to hematoxylin and eosin staining, and observed under a phase contrast microscope to determine the morphology of human ES cells and the expression of alkaline phosphatase (FIG. 1, A). The cultured cells were also assessed for telomerase activity and expression of pluripotent transcription factors (0ct4, Nanog and Sox2) using RT-PCR (FIG. 1, C) . This analysis characterized the cultured cells as being human ES cells. Also, in order to further confirm that the cultured cells are human ES cells, an immunohistochemical assay and flow cytometry through stage-specific embryonic antigen (SSEA) staining resulted in the finding that an antibody against SSEAl, a negative marker for human ES cells, does not bind to the cells, and antibodies against positive markers SSEA3 and SSEA4 bind to the cells (FIG. 1, A and B) . These results allowed the present inventors to prepare monoclonal antibodies binding specifically to undifferentiated human ES cells using embryonic stem cells themselves as an antigen.

Then, the cultured human ES cells were inactivated and used to immunize mice. Splenocytes were isolated from the mice and fused with cancer cells to generate a hybridoma. From the hybridoma, a monoclonal antibody 4-63 was isolated and purified. The monoclonal antibody was found to have binding affinity to human ES cells (FIG. 2, A) , and not to bind to mouse ES cells and mouse embryonic fibroblasts (MEFs) (FIG. 2, B) . The monoclonal antibody was also found to have decreased binding affinity to embryoid bodies differentiated from human ES cells and cells differentiated from human ES cells by retinoic acid treatment (FIG. 6, A and B) . Further, 10% SDS-PAGE analysis resulted in the finding that the monoclonal antibody 4-63 recognizes a protein of human ES cells having a molecular weight of approximately 220 kDa (FIG. 3, A) . The protein was identified as Ll cell adhesion molecule (FIG. 4; FIG. 3, B) .

In addition, the cultured human ES cells were analyzed both in an undifferentiated state and in a state of being induced to differentiate into embryoid bodies using RT-PCR for expression of transcription factors (Nanog, 0ct4 and Sox2) which are required for the maintenance of pluripotency, ectodermal (Pax6), mesodermal (CD34) and endodermal (AFP) markers, and LlCAM (FIG. 7) . Consistent with Nanog, 0ct4 and Sox2, LlCAM was expressed in the undifferentiated state of human ES cells, but displayed decreased expression in embryoid bodies (EBs) differentiated from human ES cells. These results indicated that LlCAM is a marker for the undifferentiated phenotype of human ES cells.

In order to determine whether the 4-63 antibody has application for isolation of human EScellsinan undifferentiated state, the cultured human ES cells were allowed to bind to the 4-63 antibody. The antibody-bound cells were sorted using flow cytometry, and stained with antibodies against SSEA3, which is a conventional marker for undifferentiated human ES cells, and SSEAl, which is not expressed on undifferentiated human ES cells . 98.2% of the cells sorted with the 4-63 antibody were positive for SSEA3 expression, and were negative for SSEAl expression (FIG. 8) . When human ES cells bound to anti-SSEA3 antibody were isolated and assessed for LlCAM expression, 96.8% of SSEA3-expressing cells was positive for LlCAM expression (FIG. 8) . These results indicated that LlCAM is a marker for undifferentiated human ES cells, and that the 4-63 antibody against LlCAM is very effective in isolating undifferentiated human ES cells and is thus useful therefor.

Unlike the present invention that is characterized by preparing a monoclonal antibody specific to human ES cells using human ES cells, conventional antibodies recognizing human ES cells, developed prior to the present invention, were prepared using mouse embryonic or human embryonal carcinoma cells. Such conventional antibodies include antibodies to cell-surface antigens SSEA3, SSEA4, TRA-1-60 and TRAl-1-81 (Shevinsky, etal., Cell 30:697-705, 1982; Dodd, et al., Nature 311:469-472, 1984; Andrews, etal., Hybridoma 3 : 347-361, 1984) . These conventional antibodies bind to human ES cells, but their antigens contain carbohydrates rather than proteins. The biological functions of the carbohydrate antigens have not been clearly identified. Thus, the monoclonal antibody of the present invention, binding to the human ES cell surface protein LlCAM, has antigen binding specificity obviously distinguishable from that of the conventional antibodies developed prior to the present invention.

The inventors of this application, prior to the present invention, developed monoclonal antibodies specific to human ES cell surface proteins as being disclosed in Korean Pat. Application No. 10-2004-0105717. The monoclonal antibodies 3-4B and 47-235S recognize proteins on human ES cells having respective molecular weights of approximately 26 kDa and approximately 47 kDa . The monoclonal antibody of the present invention recognizes the LlCAM antigen, which is different from those of the conventional monoclonal antibodies.

The binding specificity of the monoclonal antibody of the present invention, which is characterized by binding specifically to the human ES cell surface protein LlCAM but not binding to mouse-derived cells, demonstrates that the monoclonal antibody of this invention is a novel monoclonal antibody not having been identified prior to the present invention.

The monoclonal antibody of this invention enables the analysis of difference between undifferentiated human ES cells and mouse ES cells and mouse-derived feeder cells. Thus, the monoclonal antibody of this invention is useful in the isolation of human ES cells.

As seen from the foregoing description, the fact that the LlCAM is expressed on the surface of undifferentiated human ES cells has been not known, and an antibody against LlCAM allows high-purity isolation of undifferentiated human ES cells. Thus, the monoclonal antibody of the present invention provides a novel method of detecting, identifying and isolating undifferentiated human ES cells. The present antibody may also be used for the removal of undifferentiated human ES cells to provide differentiated functional cells for cell therapy. In another aspect , the present invention provides a hybridoma producing the monoclonal antibody 4-63, which binds specifically to the human ES cell surface protein LlCAM.

In one embodiment, the hybridoma of the present invention was prepared by irradiating human ES cells to inactivate them; intraperitoneally injecting the inactivated human ES cells into mice; isolating lymphocytes from the spleen of the mice; and fusing the lymphocytes with myeloma cells. A hybridoma secreting the monoclonal antibody 4-63 was designated as "hybridoma 4-63". The hybridoma was deposited at the Korean Collection for Type Cultures (KCTC) (Korean Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-dong, Yusong-ku, Taejon, Korea) on July 13, 2006 and assigned accession number KCTC 10966BP.

The hybridoma secreting the monoclonal antibody may be cultured in a large scale in vitro or in vivo.

The monoclonal antibody produced by the hybridoma may be used without purification, but are preferably used after being highly purified (e.g., 95% or higher) using a method known in the art in order to obtain the best results. The antibody may be isolated from culture fluid or ascites fluid using a purification technique, such as gel electrophoresis, dialysis, salting out, and chromatography.

In a detailed embodiment, for the large-scale production of the monoclonal antibody of the present invention, hybridoma cells were injected intraperitoneally into mice to be cultured in the peritoneal cavity. Ascites fluid was collected from the mice and subj ected to G-sepharose column chromatography to isolate the monoclonal antibody.

In a further aspect, the present invention provides a method of detecting or isolating undifferentiated human ES cells using an antibody binding to the LlCAM protein of human ES cells or an antibody fragment comprising an antigen-binding site thereof.

In yet another aspect, the present invention provides a method of removing undifferentiated human ES cells using an antibody binding to the LlCAM protein of human ES cells or an antibody fragment comprising an antigen-binding site thereof.

In one embodiment, the antibody specific to LlCAM or the antibody fragment comprising an antigen-binding site thereof may be used in specifically detecting, isolating or removing undifferentiated human ES cells through antigen-antibody complex formation. The LlCAM-specific antibody includes monoclonal antibodies specific to LlCAM as well as chimeric, humanized and human monoclonal antibodies. The antibody fragment comprising an antigen-binding site of the LlCAM-specific antibody is the same as described in the monoclonal antibody of the present invention. Preferably, the LlCAM-specific antibody used in the method of detecting or isolating undifferentiated human ES cells isthe4-63 monoclonal antibody according to the present invention. Antigen-antibody complex formation may be detected using histoimmunological staining, radio-immunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , Western blotting, immunoprecipitation assay, immunodiffusion assay, complement fixation assay, FACS and protein chips, but the present invention is not limited thereto.

As seen in the practice of the present invention, the LlCAM protein is a novel marker for undifferentiated human ES cells. Thus, since a substance capable of binding to the LlCAM protein, such as the monoclonal antibody of the present invention, is able to bind to LlCAM molecules on the surface of undifferentiated human ES cells, it is useful in determining the differentiated state of human ES cells, or in detecting, isolating or removing undifferentiated human ES cells.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[Mode for Invention]

EXAMPLE 1: Culture of human ES cells and characterization of the undifferentiated state of the ES cells

<1-1> Culture of human ES cells In order to prepare novel monoclonal antibodies capable of recognizing specifically human ES cells, first, human ES cell lines, Miz-hESl (Park, et al., Biol. Reprod. 69:2007-2014, 2003) and HSF6 (Abeyta, et al., Human MoI. Genet 13:601-608, 2004), were obtained from MizMedi Hospital of the Sungsam Medical Foundation. Other human ES cell lines, Hl and H9, were also obtained from the American National Institutes of Health (NIH) , and SNU-hES3 from the medical school of the Seoul National University. The human ES cells were cultured in Dulbecco's modified Eagle's medium (DMEM) /F12 (Gibco, Rockville, MD, USA) containing 20% knockout SR (Gibco) supplemented with 0.1 mM β-mercaptoethanol (Sigma, StLuis, MO, USA) , 2mMglutamine (Gibco) , 0.1 mM non-essential amino acids (Gibco), 100 U/ml penicillin G (Sigma) , 100 μg/ml streptomycin (Sigma) and 4 ng/ml bFGF (Gibco Invitrogen) , and were subcultured every 5 to 7 days.

In detail, 12-well tissue culture plates (Nunclon) were coatedwith0.1%gelatinat37^°Cforl0min. Then, gamma-irradiated

(4500 rad) mouse embryonic fibroblasts (MEF; (Animal Laboratory, Korean Research Institute of Bioscience & Biotechnology (KRIBB) ,

Korea) were seeded onto the plates at a density of 6.5χlO⁴ cells per well. The irradiated MEFs did not grow but supported the growthof human ES cells . 24 hrs after MEFculture, 5-7 day cultured human ES cell tissues were treated with 1 mg/ml of collagenase type IV (Gibco) at 37^°C for 1 hr, cut into a suitable size, and transferred to the tissue culture plates containing the irradiated MEF feeder layer. After 48 hrs, the culture medium was exchanged with a fresh medium every day.

<l-2> Evaluation of the maintenance of the cultured human ES cells in the undifferentiated state

The human ES cells cultured for 6 to 7 days in Example <1-1> were stained with hematoxylin and eosine. The human ES cells were found to grow while forming the distinct boundary with MEF feeder cells and to be closely connected with each other to form flat spherical clumps, indicating that they have characteristic morphologies of human ES cells (FIG. 1-A, panel 1) . Then, the maintenance of the undifferentiated phenotype was assessed using an alkaline phosphatase (AP) staining kit (Sigma) . The human ES cells were found to express alkaline phosphatase, indicating that they were propagated in the undifferentiated state (FIG. 1-A, panel 2 ) .

Stage-specific embryonic antigen (SSEA) staining revealed that the cultured human ES cells were negative for SSEAl as a negative control marker and positive for SSEA3 and SSEA4 as positive control markers, indicating that the cultured cells were expanded in the undifferentiated state (FIG. 1-A, panels 3, 4 and 5) . Also, In order to determine whether 0ct4 gene, not expressed in mouse embryonic fibroblasts (MEF cells) , is expressed in the human ES cells, RT-PCR was carried out with 0ct4-, Naong- and Sox2-specific primers represented by SEQ ID NOS. 1, 2 and β-actin primers represented by SEQ ID NOS. 3 and 4 for RNA guantification . Sequences of primer sets used in RT-PCR are given in Table 1, below.

TABLE 1

PCR products were then electrophoresed on a 1.5% agarose gel. The human ES cells were found to express Oct4, Nanog and Sox2, indicating that they were in the undifferentiated state (FIG. 1-B) . In FIG. 1-B, MEF indicates mouse embryonic fibroblasts, and H9, HSFβ and SNU-hES3 are human embryonic stem cells .

The human ES cells identified as being in the undifferentiated state were immunoinj ected into mice so as to prepare a hybridoma, which will be described in the following example .

EXAMPLE 2: Preparation of mouse hybridoma

Miz-hESl human ES cells cultured according to the same method as in Example <1-1> were treated with collagenase type IV. About 2xlO^β cells were suspended in 100 μl of PBS, gamma-irradiated to be inactivated, and intraperitoneally injected into Balb/cmice. Injection was repeated three times at 3-week intervals, and a final injection was carried out 3 days before cell fusion.

To prepare feeder cells, 20 ml of DMEM (GIBCO) was injected into the peritoneal cavity of healthy mice one day before cell fusion, and cells in the peritoneal cavity were suctioned and centrifuged. Splenocytes were prepared by grinding normal spleen and isolating cells from the spleen. After the feeder cells were mixed with the isolated splenocytes, the cell mixture was supplemented with 20% fetal bovine serum (FBS), plated onto a 9β-well plate at a density of 10⁵ cells per well, and cultured in a CO₂ incubator at 37 ^°C . Two weeks before cell fusion, NSl myeloma cells (ATCC TIB-18, USA) to be fused with the splenocytes were cultured in RPMI1640 medium (GIBCO) supplemented with 10% FBS.

The spleen was excised from mice immunized with human ES cells, washed with RPMI1640 medium (GIBCO), ground well in a petri dish using a glass bar, and transferred to a 15-ml tube. The tube was allowed to stand until debris precipitated. When the debris had precipitated, the supernatant was transferred to a new tube and centrifuged to recover NSl cells. The cell pellet was suspended in 10 ml of RPMI1640 medium and counted. The splenocytes were also counted. 10⁷ NSl cells were mixed with 10⁸ splenocytes in a 50-ml tube and centrifuged at 200xg for 5 min. After the supernatant was discarded, the tube was incubated in a beaker containing water at 37 ^°C for 2 min. The tube was then tapped to break up the cell pellet, and 1 ml of PEG (GIBCO) was added to the tube over one minute while the tube was gently shaken in the beaker. The cells were spun down at lOOxg for 2 min. 5 ml of RPMI1640 medium was slowly added to the tube for 3 min, and 5 ml of RPMI1640 medium was again added slowly to the tube for 2 min. After centrifugation at 200xg, the recovered cells were carefully resuspended in 30 ml of a normal medium (20% FBS-containing RPMI1640) . After being incubated in a CO₂ incubator at 37^°C for 30 min, 70 μl of the cell suspension was aliquotted onto the 96-well plate containing the MEF feeder cells at a density of 10 cells per well and cultured in a CO₂ incubator at 37 "C . The next day, 70 μl of HAT medium was added to each well, and the HAT medium was changed with a fresh one every three days for over two weeks. During this culture period, emerged colonies were observed.

Clones expressing antibodies were selected using sandwich ELISA (Enzyme-Linked Immunosorbent Assay) . 100 μl of a hybridoma culture was added to a plate coated with 2 μg/ml of an anti-mouse IgG or IgM antibody, incubated at 37 ^°C for 1 hr, and then incubated with a 1:5,000 dilution of anti-mouse IgG- or IgM-horseradish peroxidase (HRP) conjugate (Sigma) conjugate for 1 hr. The plate was washed with phosphate buffer containing 0.05% Tween 20, and a substrate solution containing OPD and H₂O₂ was added to the plate . Absorbance was measured at 492 nm to primarily select clones producing antibodies.

EXAMPLE 3: Preparation of monoclonal antibodies binding to human ES cells

<3-l> Selection of hybridoma clones producing monoclonal antibodies binding to human ES cells

Among the clones prepared in Example 2, hybridoma supernatants relatively stably secreting antibodies were evaluated for the ability to bind to human ES cells. In detail, cultured human ES cells were split using collagenase type IV and dissociated into single cells through incubation in cell dissociation buffer (GIBCO) at 37^°C for 20 min. The single-cell suspension was passed through a 40-μm cell strainer, and 2xlO⁵ cells were used for flow cytometry. First, the dissociated human ES cells were suspended in PBA (1% BSA in PBS) and allowed to react with an antibody supernatant at 4 ^°C for 30 min. The cells were centrifuged at 1200 rpm at 4°C for 5 min, and 100 μl of the antibody supernatant was discarded. Then, the cells were incubated with a 1:200 dilution of anti-mouse Ig-FITC (BD) at 4°Cfor30min. After washing with PBA twice, only propidium iodide

(PI) -negative cells were selected, and analyzed for their ability to bind to human ES cells using a FACS caliber flow cytometer.

As a result, various hybridoma clones secreting antibodies binding to human ES cells were selected and continuously subcultured for subcloning. Finally, a hybridoma clone, secreting the 4-63 antibody and reliably maintaining their stability and specificity for human ES cells, was selected.

The hybridoma secreting the monoclonal antibody 4-63 was designated as "hybridoma 4-63". This hybridoma was deposited at the Korean Collection for Type Cultures (KCTC) (KRIBB, 52, Oun-dong, Yusong-ku, Daejon, Korea) on July 13, 2006, and assigned accession number KCTC 10966BP. <3-2> Purification of the monoclonal antibody

The monoclonal antibody 4-63 was isolated from the hybridoma

4-63 selected in Example <3-l>.

In detail, to purify the 4-63 antibody, before one week, IxIO⁷ hybridoma cells were suspended in 0.5 ml of PBS and intraperitoneally injected into Balb/c mice primed with an injection with 0.5 ml of pristine. After 10 to 14 days, ascites fluid was collected using a syringe and centrifuged, and the supernatant was recovered. 1 ml of the ascites fluid was diluted with PBS to give a volume of 2 ml. The ascites fluid was then mixed with 1 nM EDTA and 0.02% NaN₃ and passed through a 0.22-μm filter. A Protein G-sepharose column (Pharmacia, Sweden) was allowed to bind to antibodies with agitation at 4°C for 2 hrs. After the column was erected vertically, the wall of the column was washed with washing buffer (0.5 M NaCl, 0.1 M Tris, pH 8.0) using a serum separator, and the column was sufficiently washed using a peristaltic pump. Then, antibody molecules were eluted with 0.2 M glycin-HCl (pH 2.7) and neutralized using 1 M Tris (pH 9.0) . The eluted antibody was dialyzed in PBS (pH 7.4) four times, aliquotted, and stored at -20^°C.

EXAMPLE 4 : Evaluation of binding specificity of the 4-63 antibody to undifferentiated human ES cells

The 4-63 antibody purified in Example <3-2> were assessed for the binding affinity to human ES cells through immunofluorescent staining according to the same method as in Example <3-l> (FIG. 2, A) . In FIG. 2-A, H9, SNU-hES3 and HSF6 are human ES cells, and the red background contains only a secondary antibody. SSEAl indicates an antibody as a negative control that does not bind to human ES cells, and SSEA3 and SSEA4 indicate antibodies as positive controls that bind to human ES cells. The blue curves indicate staining with the 4-63 monoclonal antibody, and denote that the 4-63 antibody binds to the three human ES cells.

In addition, mouse ES cells (Jl) (Li . et al ., Cell, 69 : 906-915, 1992) and mouse embryonic fibroblasts (MEF) were cultured in DMEM medium (GIBCO) supplemented with 10% FBS, and split using collagenase type IV. To determine whether the 4-63 antibody has the capacity to bind to mouse ES cells and MEF cells, according to the same method as described above, immunofluorescent staining was carried out for flow cytometry (FIG. 2, B) . The 4-63 antibody was found not to bind to Jl mouse Es cells and MEF cells.

Further, human ES cell lines, H9, SNUhES3 and HSF6, were stained with the 4-63 antibody and an antibody against TRA-1-81, which is a known cell surface marker for undifferentiated human ES cells. Both antibodies were found to co-localize on the cell surface (FIG. 2, C) . The results shown in FIG. 2 indicated that the 4-63 antibody binds to undifferentiated human ES cells.

EXAMPLE 5 : Isolation and identification of an antigen recognized by the 4-63 antibody <5-l> Immunoprecipitation assay for determining an antigen recognized by the 4-63 antibody

In order to isolate a cell surface marker on human ES cells, recognized by the 4-63 monoclonal antibody, first, a human ES cell line, SNU-hES3, was cultured, washed with PBS, and biotinylated using EZ-Link SuIfo-NHS-LC-Biotin (Pierce, Rockford, IL) . Then, the cells were lysed with lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 g/ml aprotinin, 100 g/ml phenylmethylsulfonyl fluoride, 5 g/ml leupeptin) at 4 ^°C for 20 min, and were centrifuged to remove the nuclei. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce) . Proteins nonspecifically binding to Protein G plus-Sepharose (Santa Cruz Biotechnology, Santa Cruz) were removed as follows. The cell lysate was allowed to react with 20 μl of Protein G plus-sepharose at 4 ^°C for 2 hrs and centrifuged. The supernatant was recovered and incubated with about 1 mg of the antibody at 4 ^°C for 12 hrs. The cell lysate was then mixed with 20 μl of Protein G plus-sepharose, incubated at 4 ^°C for 2 hrs, and centrifuged. The bead pellet was recovered and washed with lysis buffer ten times or more. The remaining proteins were separated on 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and subjected to Western blotting. The nitrocellulose membrane was blocked in 5% skim milk in PBST (PBS + 0.1% Tween 20) for 1 hr . After being washed with PBST twice or more, the blot was incubated with a Streptavidin-horseradish peroxidase (HRP) conjugate (1:1,500, Amersham Biosciences) for 1 hr. After being washed with PBST five times, the blot was developed using an ECL detection reagent (Amersham Biosciences) to detect biotinylated proteins. The 4-63 monoclonal antibody was found to bind to a protein having a molecular weight of about 220 kDa (FIG. 3, A) . In addition, according to the same method as described above excluding those cells that did not undergo biotinylation, H9, HSF6 and SNU-hES3 cells were lysed with the lysis buffer, immunoprecipitated with the 4-63 antibody and a commercially available anti-LICAM monoclonal antibody (5G3) . The immunoprecipitated proteins were separated on 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and subjected to Western blotting. A protein immunoprecipitated with the 4-63 antibody was detected using biotin-labeled 4-63 antibody as a primary antibody and streptavidin-HRP. As predicted, a protein of about 220 kDa was detected in the three human ES cell lines (FIG. 3, B) .

<5-2> Isolation and identification of the antigen recognized by the 4-63 antibody Immunoprecipitation was carried out with cell lysates from IxIO⁸ SNUhES3 cells according to the same method as in Example 5 in order to collect the protein immunoprecipitated with the 4-63 antibody. The immunoprecipitated protein was resolved on a 10% SDS-PAGE gel, and stained with Coomassie G250 (BioRad) according to the manufacturer's protocol. The interested protein band was excised from the gel, washed in 30% methanol for 5 min, and chopped into small pieces. The gel pieces were destained in 30% methanol . After being completely destained, the gel pieces were dehydrated in 100% acetonitrile for 10 min, and completely dried in a vacuum centrifuge for 30 min.

The dried gel pieces were incubated in a trypsin digestion solution (300 ng of trypsin (Promega), 50 mM ammonium bicarbonate) at 37°C for 16 hrs to digest proteins in gels. After the in-gel trypsin digestion was completed, the trypsin-digested peptides were extracted with 100 μl of 50 mM ammonium bicarbonate three times, and dried in a vacuum centrifuge. The peptide mixture was analyzed using a quadrupole time of flight (Q-TOF) tandem mass spectrometer having an electrospray ionization (ESI) source (Q-TOF Micro, MicroMass) . The protein recognized by the 4-63 antibody was identified as Ll cell adhesion molecule isoform 2 precursor (FIG. 4) . The underlined regions indicate the amino acid sequences analyzed through Q-TOF mass spectrometry.

In order to confirm that the 4-63 antibody binds to LlCAM, SNU-HES3 cell lysates were immunoprecipitated with a known monoclonal antibody against LlCAM, 5G3 (BD) . The immunoprecipitated proteins were separated on 10% SDS-PAGE, and subjected to Western blotting. The immunoprecipitated protein was detected using biotin-labeled 4-63 antibody as a primary antibody and streptavidin-HRP. The biotin-labeled 4-63 antibody was found to bind to the LlCAM protein immunoprecipitated with the 5G3 antibody (FIG. 3, B) . These results demonstrate that the target antigen of the 4-63 antibody is LlCAM.

EXAMPLE 6: Determination of the epitope for the 4-63 antibody on LlCAM

The LlCAM protein consists of six immunoglobulin-like domains (Ig domains) and five fibronectin type III domains. In order to determine which domain of LlCAM is responsible for binding to the 4-63 antibody, deletion mutants of LlCAM were constructed, and assessed for binding affinity to the 4-63 antibody. UJ127 and 5G3 antibodies, which are commercially available, were also used for comparison with the 4-63 antibody.

<6-l> Expression of Ll immunoglobulin domain (Ig)-Fc and Ll fibronectin type III domain (Fn)-Fc fusion proteins

In order to construct an expression vector for expressing an LlCAM immunoglobulin domain (Ig) fused to an Fc fragment of a human immunoglobulin (Ig) molecule, PCR was carried out using a plasmid DNA, pJK-dhfr2-Ll-monomer (Korean Pat. Application No. 10-2006-0079969), as a template with a pair of primers corresponding to both ends of the LlCAM Ig domain region, Ll-Igdom-F (5'-GAG GAG GAA TTC CGG CGC CGG GAA AGA TGG TCG TGG CG-3') and Ll-Igdom-R (5'-CTC CCC CTC GAG CGG CCC AGG GCT CCC CAC CAC CAA GAG CTG-3¹) . PCR conditions included denaturation at 95°C for 5 min, and 30 cycles of denaturation at 95°C for 45 sec, annealing at 58°C for 45 sec and extension at 72°C for 2 min., followed by final extension at 72°C for 10 min. A Pfu DNA polymerase (Solgent co.) was used to avoid errors in the PCR. The amplified LlCAM Ig domain DNA fragment was inserted into an expression vector, pJK-dhfr2-FC (Aprogen) , as follows. The PCR product and the expression vector were digested with EcoRI and Xhol and electrophoresed on a 1% agarose gel. The amplified DNA fragment and the linearized plasmid DNA were excised from the gel and purified using a gel purification kit (Intron co. ) . The two DNA fragments were ligated with each other using T4 DNA ligase (Roche) at lβ^°C for 12 hrs, and transfected into E. coli DH5α using a heat shockmethod. Plasmid DNA was isolated from the transformant , thereby obtaining an expression vector for the LlCAM immunoglobulin domain region, pJK-dhfr2-LlIg-Fc.

In order to construct an expression vector expressing an LlCAM fibronectin type III domain (Fn) fused to a human Ig Fc fragment, PCR was carried out using a plasmid DNA, pJK-dhfr2-Ll-monomer (Korean Pat. Application No. 10-2006-0079969), as a template with a pair of primers corresponding to both ends of the LlCAM signal peptide region, 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 ' ) , under the same conditions as described above . The amplified LlCAM signal peptide region was electrophoresed on an agarose gel and purified from the gel. In addition, the LlCAM Fn fragment was amplified through PCR, which was performed using pJK-dhfr2-Ll-monomer as a template DNA with a pair of primers corresponding to both ends of the LlCAM Fn domain region, 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') . The amplified Ll Fn fragment was then recovered. After the Ll Fn fragment was mixed with an equal amount of the Ll signal sequence fragment, PCR was carried out again using the DNA mixture as a template and a primer set of Fn-leader-F and Fn-dom-R. The amplified Ll signal sequence-Fn DNA fragment was digested with EcoRI and Xhol, and inserted into EcoRI-XhoI sites of the pJK-dhfr2-FC expression (Aprogen) , thereby obtaining pJK-dhfr2-LlFn-Fc.

In order to express Ll Ig-Fc and Ll Fn-Fc fusion proteins, pJK-dhfr2-Ll Ig-FC and pJK-dhfr2-LlFn-Fc DNA were individually transfected into HEK293T cells (ATCC No. CRL11268; hereinafter referred simply as 293T) . 20 μg of each plasmid DNA and Lipofectamine 2000 (Invitrogen) were individually mixed with 500 μl of Opti-MEM medium (Gibco BRL) , and allowed to react for 5 min at room temperature. The two solutions were combined and allowed to form DNA-Lipofectamine complexes for 15 min at room temperature. The DNA/Lipofectamine mixture was mixed with 4 ml of Opti-MEM medium, and carefully overlaid onto 293T cells. The cells were incubated in an incubator at 37 ^°C under 5% CO2. After 6 hrs, the cells were refed with 5 ml of 10% FBS-containing DMEM and cultured for a further 3 days.

<6-2> Western blotting The 293T cell cultures expressingLl Ig-FC andLl Fn-Fc fusion proteins were subjected to 7.5% SDS-PAGE to separate proteins. The separated proteins were transferred onto a nitrocellulose membrane. The blot was incubated in the 4-63 antibody or a known antibody, UJ127 (Chemicon) or 5G3 (Pharmingen) , for 1 hr, and washed with TBST buffer (TBS + 0.05% Tween 20) three times. The blot was then incubated in anti-mouse IgG-HRP conjugate ( (1:5000 diluted, Sigma) for 1 hr. The blot was washed with TBST buffer five times, and developed with an ECL detection reagent (Amersham Biosciences) . The 4-63 antibody and the known antibody 5G3 were found to bind the Ig-Fc construct of about 140 kDa, and the known antibody UJ127 was found to bind to the Fn-Fc construct of about 140 kDa (FIG. 5, A) .

<6-3> Preparation of deletion mutants of LlCAM Ig domains and determination of the epitope for the 4-63 antibody on LlCAM In order to construct expression vectors expressing domains 1 to 5 (1-5) , domains 1 to 4 (1-4) , domains 1 to 3 (1-3) , domains 1 to 2 (1-2) , and only domain 1 (1) among six LlCAM Ig domains, PCR was carried out using pJK-dhfr2-LHg-Fc as a template DNA with a pair of primers, 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-3' ) , Ll-Ig3dom-R (5'-CTC CCC CTC GAG CTC CAC GGT GAC ATA GTA CGC-3' ) , Ll-Ig2dom-R (5'-CTC CCC CTC GAG CTT GAC CCG GAG GTC AAT GGG-3¹) or Ll-Igldom-R (5'-CTC CCC CTCGAGCTCGGCCATGAGCCGGATCTC-S') . The amplified DNA fragments were digested with EcoRI and Xhol, and inserted into EcoRI/XhoI sites of the pJK-dhfr2-Fc expression vector (Aprogen) , thereby obtaining pJK-dhfr2-LlIgl-5dom-Fc, pJK-dhfr2-LlIgl-4dom-Fc, pJK-dhfr2-LlIgl-3dom-Fc, pJK-dhfr2-LlIgl-2dom-Fc, and pJK-dhfr2-LUgldom-Fc, respectively.

Then, the expressed vectors were transfected into 293T cells and allowed to express Fc fusion proteins of the deletion mutants of LlCAM Ig domains, Ll Igl-5, Igl-4, Igl-3, Igl-2 and IgI. Each cell culture was subjected to Western blot analysis with 4-63 and 5G3 antibodies. The 4-63 antibody was found to bind to all Ig-Fc fusion proteins with IgI, Igl-2, Igl-3, Igl-4 and Igl-5, indicating that the 4-63 antibody binds to the first Ig domain (FIG. 5, B) . The 5G3 antibody was found not to bind to IgI but to bind to Igl-5, Igl-4, Igl-3 and Igl-2 (FIG. 5, C) . These results indicated that the 4-63 antibody binds to an epitope different from that of UJ127 and 5G3 antibodies.

EXAMPLE 7: Evaluation of LlCAM expression on the surface of undifferentiated human ES cells

Human ES cells were detached with collagenase, and transferred to a bacterial culture plate with caution being made not to break up cell clumps, and cultured in an embryoid body

(EB) growth medium (Dulbecco' s modified Eagle' s medium (DMEM) /Fl2

(Gibco) supplemented with 20% FBS (Hyclone) , 0.1 mM β-mercaptoethanol (Sigma), 2 mM glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco) , 100 U/ml penicillin G (Sigma) , and 100 μg/ml streptomycin (Sigma)) for 4 days so as to allow EB formation. The medium and the plate were changed with a fresh one every day. The human ES cells differentiated embryoid bodies were subjected to FACS analysis with the 4-63 antibody according to the same method as in Example 3-1. The differentiated human ES cells exhibited a sharp decrease in SSEA3 and SSEA4 expression. Consistent with the decreased expression of SSEA3 and SSEA4, the expression of LlCAM, recognized by the 4-63 antibody, was also sharply reduced (FIG. 6, A) . When exposed to retinoic acid, human ES cells tend to lose the ability to maintain their undifferentiated state and to differentiate (Henderson, et al., Stem Cells 20 : 329-337 , 2002) . H9 cells cultured for 4 days were either treated with 10^~5 M of retinoic acid for 8 days or not treated, detached, and subjected to FACS analysis with the above antibodies according to the same method as in Example 3-1 (FIG. 6, B) . The 4-63 antibody showed decreased binding affinity to the differentiated cells. These results indicated that the expression of LlCAM recognized by the 4-63 antibody is specific to undifferentiated human ES cells . In addition, the cultured human ES cells were analyzed both in an undifferentiated state and in a state of being induced to differentiate into embryoidbodies using RT-PCR for expression of pluripotent transcription factors, Nanog, Oct4 and Sox2, and ectodermal (Paxδ), mesodermal (CD34) and endodermal (AFP) markers, and LlCAM (FIG. 7) . Total RNA was isolated from H9, HSF6 and SNU-hES3 cells and embryoid bodies induced from each cell line (EB on Day 6 and Day 12) using a TriZol reagent. 5 μg of total RNA cDNA was used for cDNA synthesis. RT-PCR was carried out using a RT-PCR kit (Superscript™ III first-strand synthesis system for RT-PCR, Invitrogen) with the primers specific to markers for undifferentiated and differentiated phenotypes, listed in Table 1. Consistent with Nanog, 0ct4 and Sox2, LlCAM was expressed in the undifferentiated state of human ES cells, but displayed decreased expression in embryoid bodies (EBs) differentiated from human ES cells. These results indicated that LlCAM is a marker for the undifferentiated phenotype of human ES cells.

EXAMPLE 8: Isolation of undifferentiated human ES cells using the 4-63 antibody

In order to determine whether the 4-63 antibody is able to selectively isolate undifferentiated human ES cells, a human ES cell line, H9, was cultured and stained with the 4-63 antibody. The antibody-bound cells were isolated using a BD FACSAria cell sorter, and assessed for their binding specificity to antibodies against SSEA3, which is a conventional marker for the undifferentiated phenotype, and SSEAl, which is a conventional marker for the differentiated phenotype. 98.2% of the cells isolated with the 4-63 antibody expressed SSEA3 but did not express SSEAl (FIG. 8) . In addition, when human ES cells bound to anti-SSEA3 antibody were isolated and assessed for LlCAM expression, 96.8% of SSEA3-expressing cells was positive for LlCAM expression (FIG. 8) . These results indicated that LlCAM isamarkerforthe undifferentiated phenotype of human ES cells, and that the 4-63 antibody specific to LlCAM is useful in identifying and isolating undifferentiated human ES cells.

[industrial Applicability]

As described hereinbefore, the monoclonal antibody of the present invention binds specifically to LlCAM, which is expressed on undifferentiated human ES cells . Thus, the monoclonal antibody is very useful in identifying and isolating undifferentiated human

ES cells. As well, the monoclonal antibody is useful for the removal of undifferentiated human ES cells from their differentiated progeny in cell therapy.

Claims

[CLAIMS] [Claim l]

A method of detecting or isolating undifferentiated human embryonic stem cells using an antibody binding to Ll cell adhesion molecule (LlCAM) on human embryonic stem cells, or an antibody fragment comprising an antigen-binding site thereof.

[Claim 2]

The method according to claim 1, wherein the antibody fragment is Fab, F(ab'), F(ab')₂ or Fv.

[Claim 3]

The method according to claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody and a human monoclonal antibody.

[Claim 4]

The method according to claim 3, wherein the antibody is a monoclonal antibody (4-63) produced by a hybridoma having accession number KCTC 10966BP.

[Claim 5] A hybridoma assigned accession number KCTC 10966BP.

[Claim 6]

A monoclonal antibody (4-63) which is produced by the hybridoma of claim 5.

[Claim 7] A method of removing undifferentiated human ES cells using an antibody specific to Ll cell adhesion molecule (LlCAM) or an antibody fragment comprising an antigen-binding site thereof.

[Claim 8]

The method according to claim 7, wherein the antibody against LlCAM is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody and a human monoclonal antibody.

[Claim 9]

The method according to claim 7 , wherein the antibody fragment is Fab, F(ab'), F(ab')2 or Fv.

[Claim 10]

The method according to claim 7, wherein the antibody is a monoclonal antibody (4-63) produced by a hybridoma having accession number KCTC 10966BP.