Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0081—Purging biological preparations of unwanted cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies
  • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/16—Animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells

Definitions

• the present invention relates to monoclonal antibodies that specifically bind to cell surface proteins of human embryonic stem cells and hybridomas producing the same.
• Stem cells have the ability to differentiate into a variety of cell types and they collectively mean undifferentiated cells prior to the differentiation that can be obtained from embryo, fetus, and adult body tissues.
• stem cells Upon appropriate stimulus or in environment, stem cells are capable to differentiate into certain specific cell types. Unlike differentiated cells where cell division is stopped, stem cells can multiply by division, the process being called self-renewal or proliferation. Stem cells also exhibit plasticity and they are capable of differentiating into different cell types upon exposure to different environments or stimuli. Stem cells are typically divided into two groups, pluripotent embryonic stem cells and multipotent adult stem cells . Embryonic stem cells are derived from the inner cell mass which will form the future fetus within the blastocyst during early embryogenesis, and have the potential to differentiate into every cell type of every tissue comprising an individual.
• tissue-specific stem cells primary stem cells
• the tissue-specific stem cells are described as multipotent or unipotent because they usually have a restricted capacity for differentiation into cells specific to the tissue in which they are found. In adults, the tissue-specific stem cells remain in most tissues and replenish normally or pathologically lost cells.
• embryonic stem cells Since embryonic stem cells have a special quality of pluripotency (the ability to develop into all cell types) , they may be 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. Thus, embryonic stem cells are receiving increasing attention as an effective treatment of various incurable diseases.
• Human embryonic stem (ES) cells share some features in common with mouse embryonic stem (ES) cells, most of which have been well identified, but are known to have many distinct differences from mouse ES cells. First, representative similarities are as follows.
• human ES cells are pluripotent like mouse ES cells, they differentiate into various cell types of all tissues when induced to form embryoid bodies (EBs) in vitro (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).
• Human and mouse embryonic stem cells can both be cultivated in the presence of feeder layers or conditioned media from feeder layers.
• human ES cells like mouse ES cells, express Oct-4, which is known to participate in early-stage differentiation during embryonic development, show telomerase activity, which is expressed in cells having self- proliferative ability, and express alkaline phosphatase, which is expressed in high levels in mouse ES cells.
• these human ES cells also exhibit marked differences from their murine counterparts, as follows.
• Mouse ES cells can be cultivated using a single cell, but human embryonic stem cells are impossible to cultivate from a single cell because they are mostly dead when a single cell is used.
• Human and mouse ES cells are also different from each other in morphology and cytokine requirement for maintaining self- reproduction or pluripotency during cultivation.
• stem cell-based therapies include isolation of stem cells from humans and animals, establishment of stem cell cultivation techniques, induction of differentiation into specific functional cells under in vitro conditions and isolation of differentiated cells, insurance of efficacy and safety in animals before clinical trials, and techniques of suppressing the immune rejection of stem cell transplants in the body.
• the most important technique involves inducing differentiation into various specialized cells.
• FIG. la photographically shows the morphology of cultured human ES cells (1), and the results of the analysis of expression of ES cell surface markers: alkaline phosphatase (2), SSEAl (3), as a negative control marker, and SSEA3 (4) and SSEA4 (5), as positive control markers, wherein the expression of SSEAl, SSEA3 and SSEA4 is measured by an immunohistochemical assay;
• lb shows the results of electrophoresis of products of RT-PCR (Reverse transcriptase-Polymerase Chain Reaction) carried out to determine whether 0CT4 (octamer binding protein 4) gene, not expressed in mouse embryonic fibroblasts (MEF) , is expressed in human ES cells (-RT: negative control not containing reverse transcriptase, MEF: mouse embryonic fibroblasts, and hES: human embryonic stem cells) ;
• RT-PCR Reverse transcriptase-Polymerase Chain Reaction
• lc shows the results of Southern blotting, displaying that MEF cells do not express telomerase and that human ES cells express heat-sensitive telomerase that leads to an increase in telomere length (P: positive control extract and its serial diluents, P+heat: inactivated telomerase by heat treatment of positive control (sample) , MEF: extract of mouse embryonic fibroblasts, hES: extract of human embryonic stem cells, and hES+heat: heat-treated extract of human ES cells) ; FIG.
• FIG. 2 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention bind to Miz- hESl and HSF6 human ES cells, wherein the solid line represents a monoclonal antibody, and the red background contains only a secondary antibody (SSEAl: antibody not binding to human ES cells (negative control) , SSEA3 and SSEA : antibodies binding to human ES cells (positive controls) ;
• SSEAl antibody not binding to human ES cells (negative control)
• SSEA3 and SSEA antibodies binding to human ES cells (positive controls)
• FIG. 3 shows the results of immunocytostaining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention bind to Miz-hESl and Miz- hES4 human ES cells (SSEAl: antibody not binding to human ES cells (negative control), SSEA3 and SSEA4 : antibodies binding to human ES cells (positive controls) ;
• SSEAl antibody not binding to human ES cells (negative control)
• SSEA3 and SSEA4 antibodies binding to human ES cells (positive controls) ;
• FIG. 4 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention do not bind to mouse ES cells, wherein the solid line represents a monoclonal antibody, and the red background contains only a secondary antibody (SSEA4: antibody not binding to mouse ES cells (negative control), SSEAl: antibody binding to mouse ES cells (positive control) ;
• FIG. 5 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention do not bind to mouse embryonic fibroblasts, wherein the solid line represents a monoclonal antibody, and the red background contains only a secondary antibody;
• FIG. 4 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention do not bind to mouse ES cells, wherein the solid line represents a monoclonal antibody, and the red background contains only a secondary antibody;
• FIG. 6 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention do not bind to mouse STO fibroblasts, used as supporting cells in the culture of human ES cells, wherein the solid line represents a monoclonal antibody, and the red background contains only a secondary antibody;
• FIG. 7 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention do not bind to human neural progenitor cells, hNPSTl, differentiated from human ES cells, wherein the solid line represents a monoclonal antibody, and the red background contains only a secondary antibody;
• FIG. 7 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention do not bind to human neural progenitor cells, hNPSTl, differentiated from human ES cells, wherein the solid line represents a monoclonal antibody, and the red background
• FIG. 8 shows the results of fluorescent cell staining, displaying that 3-4B and 47-235S monoclonal antibodies according to the present invention have reduced binding affinity to embryonic stem cells in the presence of retinoic acid inducing the differentiation of human ES cells, wherein the expression of SSEAl, as a negative control, and SSEA3 and SSEA4, as positive controls, is also assessed;
• FIG. 9 shows the results of an immunoprecipitation assay for biotinylated human ES cells with 3-4B and 47-235S monoclonal antibodies according to the present invention, wherein precipitated proteins are assessed by SDS-PAGE followed by Western blotting, and bands in the lower panel represent heavy and light chains of antibodies used (negative control: tested under the same conditions except for not using the antibody) ; and FIG. 10 shows the results of Western blotting for binding affinity of a 47-235S monoclonal antibody according to the • present invention to various cell types ( ⁇ -actin is used as an internal control for protein concentrations) .
• the present invention relates to a monoclonal antibody that binds to a cell surface protein of human embryonic stem (ES) cells but does not bind to mouse embryonic stem (ES) cells.
• the monoclonal antibody of the present invention binds to undifferentiated human ES cells but does not bind to differentiated human ES cells.
• the monoclonal antibody of the present invention specifically binds to a protein of human ES cells having a molecular weight of about 26 kDa on 10% SDS-PAGE.
• the monoclonal antibody of the present invention specifically binds to a protein of human ES cells having a molecular weight of about 47 kDa on 10% SDS-PAGE.
• the monoclonal antibody of the present invention is a 3-4B monoclonal antibody that is produced by a hybridoma assigned accession number KCTC 10599BP.
• the monoclonal antibody of the present invention is a 47-235S monoclonal antibody that is produced by a hybridoma assigned accession number KCTC 10739BP.
• the term "monoclonal antibody”, as used herein, refers to a protein molecule that is directed by a single antigenic region (single epitope) and specifically binds thereto. With respect to the objects of the present invention, since the monoclonal antibody of the present invention specifically binds to a cell surface protein of human ES cells in an undifferentiated state, the monoclonal antibody is a protein molecule recognizing a cell surface protein of undifferentiated human ES cells.
• the major regions of an antibody involved in the recognition of a specific epitope and the formation of antigen-antibody complexes are variable regions of heavy chain and light chain, and in particular, CDR (complementary determining region) attributes to the formation of antigen-antibody complexes.
• the present invention includes chimeric antibodies and humanized antibodies of the monoclonal antibody, which comprise variable regions of the monoclonal antibody of the present invention, especially CDR.
• the present invention further 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 features.
• “Functional fragments of antibody molecules” mean fragments retaining at least an antigen-binding function, and include Fab,
• F(ab') , F(ab') 2 and Fv 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 features of the human ES cells, confirmed that the cultured cells are human ES cells, and immunoinjected the cultured human ES cells into mice.
• human ES cells were cultured, subjected to hematoxylin and eosin staining, observed under a phase contrast microscope to determine the morphology of human ES cells and the expression of alkaline phosphatase (see, panels 1 and 2 of FIG.
• telomerase activity see, FIG. lc
• 0ct4 expression see, FIG. lb
• SSEA stage-specific embryonic antigen
• ES cells mouse embryonic fibroblasts and mouse fibroblasts (STO) (FIGS. 4 to 6) .
• the monoclonal antibodies were found to have decreased binding affinity to hNPSTl neural progenitor cells (Korean Pat. Application No. 10-2004- 0011705) differentiated from human ES cells and cells differentiated from human ES cells by retinoic acid treatment (FIGS. 7 and 8) .
• hNPSTl neural progenitor cells Kerean Pat. Application No. 10-2004- 0011705
• retinoic acid treatment retinoic acid treatment
• 10% SDS-PAGE analysis resulted in the finding that the monoclonal antibodies 3-4B and 47-235S recognize proteins of human ES cells having molecular weights of about 26 kDa and about 47 kDa, respectively (FIG. 9) .
• the molecular weights of the human ES cell proteins, recognized by the monoclonal antibodies of the present invention are determined by 10% SDS-PAGE analysis, they may increase or decrease within a certain range according to measurement conditions.
• the term "about” is unavoidably used to express the molecular weights of the proteins, and is typically within a range of ⁇ 2 kDa, preferably +1 kDa.
• conventional antibodies recognizing human ES cells, developed prior to the present invention were prepared using mouse embryonic or human embryonal carcinoma cells.
• Conventional antibodies include antibodies to cell-surface antigens SSEA3, SSEA4, TRA-1- ⁇ 60 and TRA1-1- 81 (Shevinsky, et al., Cell 30:697-705, 1982; Dodd, et al., Nature 311:469-472, 1984; Andrews, et al., Hybridoma 3:347- 361, 1984).
• SSEA3, SSEA4, TRA-1- ⁇ 60 and TRA1-1- 81 Shevinsky, et al., Cell 30:697-705, 1982; Dodd, et al., Nature 311:469-472, 1984; Andrews, et al., Hybridoma 3:347- 361, 1984.
• SSEA3, SSEA4, TRA-1- ⁇ 60 and TRA1-1- 81 Shevinsky, et al., Cell 30:697-705, 1982; Dodd, et al., Nature 311:469-472, 1984; Andrews, et al
• an anti-CD9 antibody is known to recognize a cell surface protein of human ES cells, but it binds to mouse ES cells as well as human ES cells (Oka, et al., Mol. Biol. Cell 13:1274-1281, 2002; Carpenter, et al., Dev. Dyn. 229:243-258, 2004).
• This antigen binding specificity of the anti-CD9 antibody is obviously distinguished from the binding specificity of the monoclonal antibodies of the present invention only to human ES cells. That is, the anti ⁇ CD9 antibody recognizes an antigenic protein present in both human and mouse ES cells. In contrast, the monoclonal antibodies of the present invention recognize antigenic proteins present only in human ES cells.
• Some antibodies against ES cells can selectively bind to ES cells that originate from different species, such as humans and mice.
• mouse ES cells bind to an anti-SSEAl antibody but not to antibodies against SSEA3 and SSEA4.
• human ES cells bind to anti-SSEA3 and anti-SSEA4 antibodies but not to an anti-SSEAl antibody (Thomson et al . , Science, 282: 1145-1147, 1998).
• antibodies against TRA-1-60 and TRA-1-81 derived from human embryonal carcinoma cells do not bind to mouse ES cells but bind to human ES cells (Andrews, et al., Hybridoma 3:347-361, 1984).
• the binding specificity of the monoclonal antibodies of the present invention which is characterized by specifically binding to cell surface proteins of human ES cells but not binding to mouse ES cells, demonstrates that the monoclonal antibodies of the present invention are novel monoclonal antibodies not identified prior to the present invention.
• the monoclonal antibody of the present invention since the monoclonal antibody of the present invention, 47-235S, recognizes both a normal form of a protein antigen (conformational protein .epitope) and a denatured form of the protein antigen (linear protein epitope) , it may be more useful for the analysis of biological functions of human ES cells .
• the present invention relates to two hybridomas producing the monoclonal antibodies of the present invention.
• the present invention provides two hybridomas producing the monoclonal antibodies 3-4B and 47-235S.
• the hybridomas of the present invention were 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.
• hybridoma secreting a monoclonal antibody 3-4B was designated as "hybridoma 3-4B", which was deposited at KCTC (Korean Collection for Type Cultures, Genetic Resources Center, KRIBB, 52, Oun-dong, Yusong-ku, Taejon, Korea) on Feb. 17, 2004 and assigned accession number KCTC 10599BP.
• KCTC Korean Collection for Type Cultures, Genetic Resources Center, KRIBB, 52, Oun-dong, Yusong-ku, Taejon, Korea
• the hybridomas secreting monoclonal antibodies may be cultured in a large scale in vitro or in vivo.
• the monoclonal antibodies secreted by the hybridomas may be used without purification, but are preferably used after being highly purified (e.g., 95% or higher) by methods known in the art in order to obtain the best results. Purification may be carried out using culture fluid or ascites fluid, for example, using gel electrophoresis, dialysis, salting out and chromatography.
• hybridoma cells were intraperitoneally injected into mice to be cultured in the peritoneal cavity, and ascites fluid was collected from the mice and subjected to Protein G-sepharose column chromatography to isolate the monoclonal antibodies .
• the present invention relates to a composition for removing undifferentiated human ES cells comprising the monoclonal antibody.
• the present invention relates to a method of removing undifferentiated human ES cells using the monoclonal antibody.
• the monoclonal antibodies of the present invention may be used for removing ES cells present in cells to be transplanted for cell therapies or in transplanted cells.
• the monoclonal antibodies of the present invention may be linked to a known therapeutic agent by direct or indirect coupling (e.g., covalent bonding) through a linker.
• therapeutic agents capable of being linked to the antibodies include radionuclides, drugs, lymphokines, toxins and heterologous antibodies.
• the antibodies may be administered as ther are, or compositions comprising the antibodies may be administered.
• the compositions comprising the antibodies may include an acceptable carrier according to administration methods and may be formulated into suitable pharmaceutical preparations . Suitable pharmaceutical preparations according to administration methods are known in the art.
• compositions comprising the antibodies of the present invention may be administered in an amount pharmaceutically effective for removing ES cells . Typical dosage levels may be optimized using a standard clinical technique .
• the present invention relates to assay kits for undifferentiated human ES cells comprising the monoclonal antibodies.
• the monoclonal antibodies of the present invention may be used for specifically detecting undifferentiated human ES cells through an antigen-antibody complex reaction, as well as removing embryonic stem (ES) cells from cells to be transplanted or transplanted cells.
• these assay kits may include tools and reagents, which are generally used in the art for immunological analysis. These tools/reagents include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, solubilizing agents, detergents, buffering agents, and stabilizing agents.
• the assay kit may include a substrate allowing the measurement of enzyme activity and a reaction terminator.
• Suitable carriers include, but are not limited to, soluble carriers, for example, physiologically acceptable buffers known in the art, for example, PBS, insoluble carriers, for example polymers such as polystylene, polyethylene, polypropylene, polyesters, polyacrylnitrile, fluorocarbon resin, crosslinked dextran, polysaccharides and magnetic microparticles composed of latex plated with metals, papers, glasses, metals, agarose, and combinations thereof.
• Antigen-antibody complex formation may be detected by 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 to these examples .
• Labels allowing qualitative or quantitative analysis of the formation of antigen-antibody complexes include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive isotopes .
• enzymes available as detection labels include, but are not limited to, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urase, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphenolpyruvate decarboxylase, and ⁇ -latamase.
• fluorescent substances include, but are not limited to, fluorescin, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamin.
• ligands include, but are not limited to, biotin derivatives.
• luminescent substances include acridinium esters, luciferin and luciferase.
• the microparticles include, but are not limited to, colloidal gold and colored latex.
• redox molecules examples include, but are not limited to, ferrocene, ruthenium complexes, viologen, quinone, Ti ions, Cs ions, diimide, 1, 4-benzoquinone, hydroquinone, K 4 W(CN) 8 , [Os(bpy) 3 ] 2+ . [RU(bpy) 3 ] 2+ , and [MO(CN) 8 ] 4" .
• radioactive isotopes include, but are not limited to, 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re.
• EXAMPLE 1 Culture of human ES cells and analysis of their features
• the human ES cells were cultured in DMEM (Dulbecco's modified Eagle's medium) /F12 (Gibco, Rockville, MD, USA) containing 20% knockout SR (Gibco) supplemented with 0.1 mM ⁇ - mercaptoethanol (Sigma, St Luis, MO, USA), 2 mM glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 100 U/ml penicillin G (Sigma), 100 ⁇ g/ml streptomycin (Sigma), and 4 ng/ml bFGF (Gibco Invitrogen) , and were subcultured every 6 days .
• DMEM Dynabecco's modified Eagle's medium
• F12 Gibco, Rockville, MD, USA
• 20% knockout SR Gibco
• 0.1 mM ⁇ - mercaptoethanol Sigma, St Luis, MO, USA
• 2 mM glutamine Gibco
• Hematoxylin was completely removed using ammonia water, and the cells were treated with 0.5% eosine (Sigma) for 5 min. After being washed with ammonia water, the cells were treated sequentially with 70%, 80%, 95% and 100% ethanol for 1 min for each treatment and observed by phase contrast microscopy (the panel 1 of FIG. la) .
• the human ES cells grew while forming the distinct boundary with MEF feeder cells and were closely connected with each other to form flat spherical clumps, indicating that they have characteristic morphologies of human ES cells.
• ⁇ l-4> SSEA staining Human ES cells cultured according to the same method as in the ⁇ 1-1> of Example 1 were fixed with 4% paraformaldehyde for 30 min. After being washed with PBS, the cells were blocked in typical horse serum for 1 hr. Subsequently, the cells were incubated with antibodies to stage specific embryonic antigen (SSEA) 1, SSEA3 and SSEA4 (DSHB, the University of Iowa, USA) for 1 hr. After being washed with PBS, the cells were incubated with a secondary antibody to SSEA for 1 hr.
• SSEA stage specific embryonic antigen
• the cells were stained using Vectastain ABC reagents (DAP staining kit, Sigma) for 20 min, washed with PBS and treated with a substrate solution. The cells were immunohistochemically observed under a phase contrast microscope.
• the human ES cells were negative for SSEAl as a negative control marker and positive for SSEA3 and SSEA4 as positive control markers (the panels 3, 4 and 5 of FIG. la) .
• telomere activity assay kit (Intergen, NY, USA) according to the manufacturer's protocol.
• a cell lysate was mixed with a TS primer, and telomerase in the cell lysate was allowed to elongate the TS primer. Then, PCR was carried out using an RP primer and Taq polymerase . PCR conditions included 36 cycles of 94°C for 30 sec, 59°C for 30 sec and 72°C for 1 min. PCR products were electrophoresed on a 2% agarose gel and transferred onto a membrane. Southern blotting was carried out using a 32 P-labeled-primer having the oligonucleotide sequence represented by SEQ ID NO. 5 to measure telomerase activity. The human ES cells were found to express heat-sensitive telomerase (FIG. lc) . In FIG.
• the lanes represent the following: P: positive control extract and its diluents, P+heat: telomerase inactivated by heat treatment of positive control (sample) , MEF: extract of mouse embryonic fibroblasts, hES: extract of human embryonic stem cells, and hES+heat: heat-treated extract of human ES cells .
• RNA isolation kit (Roche) .
• RT-PCR was carried out with 0ct4- specific primers represented by SEQ ID NOS. 1 and 2 and ⁇ - actin primers represented by SEQ ID NOS. 3 and 4 for RNA quantification. Then, PCR products were electrophoresed on a 1.5% agarose gel .
• the human ES cells were found to express Oct4 (FIG. lb) .
• the lanes represent the following: -RT: negative control not containing reverse transcriptase, MEF: mouse embryonic fibroblasts, and hES: human embryonic stem cells.
• NS1 myeloma cells (ATCC, USA) to be fused with the splenocytes were cultured in a 10% FBS-containing medium.
• the spleen was excised from mice immunized with human ES cells according to the same method as in the ⁇ 2-l> of Example 2, washed with RPMI1640 (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 NS1 cells. The cell pellet was suspended in 10 ml of RPMI1640 and counted.
• the splenocytes were also counted. 10 7 NS1 cells were mixed with 10 8 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° 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 was slowly added to the tube for 3 min, and 5 ml of RPMI1640 was again added slowly to the tube for 2 min.
• PEG GABA
• the recovered cells were carefully resuspended in 30 ml of a normal medium (RPMI1640 +20% FBS) . After being incubated in a C0 2 incubator at 37° for 30 min, 70 ⁇ l of the cell suspension was aliquotted onto the 96-well plate containing the MEF feeder cells at a density of 10 5 cells per well and cultured in a C0 2 incubator at 37°. The next day, 70 ⁇ l of HAT was added to each well, and the HAT medium was changed 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) .
• a hybridoma culture 100 ⁇ l of a hybridoma culture was added to a plate coated with 2 ⁇ g/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 an anti-mouse IgG or IgM HRP (horseradish peroxidase, Sigma) conjugate for 1 hr.
• the plate was washed with phosphate buffer containing 0.05% Tween 20, and a substrate solution containing OPD and H 2 0 2 was added to the plate. Absorbance was measured at 492 nm to primarily select clones producing antibodies .
• hybridoma supernatants relatively stably secreting antibodies were evaluated for the ability to bind to human ES cells.
• cultured human ES cells were split using collagenase type IV and dissociated into single cells by incubation with cell dissociation buffer (GIBCO) at 37°C for 20 min.
• the single-cell suspension was passed through a 40- ⁇ m cell strainer, and 2xl0 5 cells were used for flow cytometry.
• 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 ⁇ l of the antibody supernatant was discarded. Then, the cells were incubated with a 1:200 dilution of anti-mouse Ig-FITC (BD) at 4°C for 30 min. 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.
• BD anti-mouse Ig-FITC
• hybridoma secreting a monoclonal antibody 3-4B was designated as "hybridoma 3-4B", deposited at KCTC (Korean Collection for Type Cultures, Genetic Resources
• hybridoma secreting a monoclonal antibody 47-235S was designated as "hybridoma 47-235S", deposited at KCTC on Dec. 6, 2004, and assigned accession number KCTC 10739BP.
• Monoclonal antibodies 3-4B and 47-235S were isolated from the hybridomas 3-4B and 47-235S, respectively, selected in the ⁇ 3-l> of Example 3.
• lxlO 7 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% NaN 3 and passed through a 0.22- ⁇ m filter.
• a Protein G-sepharose column (Pharmacia, Sweden) was allowed to bind to antibodies by rotation at 4°C for 2 hrs. After the column was stood up 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, antibodies were eluted with 0.2 M glycin-HCl (pH 2.7) and neutralized using 1 M Tris (pH 9.0). The purified 3-4B and 47-235S antibodies were used in the following Examples.
• the 3-4B and 47-235S antibodies purified in the ⁇ 3-2> of Example 3 were assessed for the binding affinity to human ES cells by fluorescent cell staining according to the same method as in the ⁇ 3-l> of Example 3 (FIG. 2) .
• the solid line represents a monoclonal antibody, 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
• SSEA 4 indicates an antibody as a positive control that binds to human ES cells .
• Miz-hESl and Miz-hES4 human ES cells were fixed with 4% formaldehyde for 30 min, and were analyzed using an immunocytochemical assay with antibodies specific to SSEAl, SSEA4, 3-4B and 47-235S.
• SSEAl as a negative -control marker
• SSEA3 and SSEA 4 as positive control markers
• the human ES cells were negative for SSEAl and positive for SSEA 3 and SSEA 4 (FIG. 3) .
• the monoclonal antibodies 3-4B and 47- 235S all were found to bind to both Miz-hESl and Miz-hES4 human ES cells (FIG. 3) .
• mouse ES cells Jl
• mouse embryonic fibroblasts MEF
• mouse STO fibroblasts ATCC 56-X
• 235S antibodies have the capacity to bind to mouse ES cells, MEF cells and mouse STO fibroblasts, according to the same method as described above, fluorescent cell staining was carried out for flow cytometry (FIGS. 4, 5 and 6) .
• the 3-4B and 47-235S antibodies were found not to bind to the above cell lines.
• the 3-4B and 47-235S antibodies were found not to bind to human neural progenitor cells (hNPSTl) differentiated from human ES cells (FIG. 7) .
• EXAMPLE 5 Evaluation of the binding affinity of the monoclonal antibodies to differentiated human ES cells
• Miz-hESl cells were treated with 10 ⁇ 5 M retinoic acid for 6 days, and additional cells were not treated. Then, the cells were detached and subjected to FACS analysis using the monoclonal antibodies according to the same method as in the ⁇ 3-l> of Example 3 (FIG. 8) .
• the monoclonal antibodies exhibited significantly reduced binding affinity to the differentiated cells, indicating that the monoclonal antibodies of the present invention specifically bind to undifferentiated human ES cells.
• EXAMPLE 6 Immunoprecipitation assay for determining antigens recognized by the monoclonal antibodies
• cultured human ES cells were washed with PBS and biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) .
• 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 BCA (bicinchoninic acid) protein assay kit (Pierce) . Proteins nonspecifically binding to Protein G plus-Sepharose (Santa)
• the cell lysate was allowed to react with 20 ⁇ l of Protein G plus-sepharose at 4°C for 2 hrs and centrifuged. The supernatant was recovered and incubated with about 1 ⁇ g of an antibody at 4°C for 12 hrs. The cell lysate was then mixed with 20 ⁇ l of Protein G plus-sepharose, incubated at 4°C for 2 hrs and centrifuged. The 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.
• the blot was incubated with a Streptavidin-HRP (horseradish peroxidase) conjugate (1:1,500, Amersham Biosciences) for 1 hr. After being washed with PBST five times, the blot was developed using ECL detection reagents (Amersham Biosciences) to detect biotinylated proteins (FIG. 9) .
• the 3-4B monoclonal antibody was found to bind to a protein having a molecular weight of about 26 kDa, and the 47-235S monoclonal antibody to a protein having a molecular weight of about 47 kDa.
• EXAMPLE 7 Analysis of the 47-235S antibody by Western blotting
• Example 6 In order to determine whether the antibody prepared in the above Example is able to recognize its antigen even in a denatured state, 10% SDS-PAGE was carried out using cell lysates of STO, MEF, mESC(Jl), Miz-hESl and hNPSTl cells. Western blotting was carried ' out using a nitrocellulose membrane according to the same method as in Example 6.
• the nitrocellulose membrane was blocked in 5% skim milk in PBST (PBS + 0.1% Tween 20) for 1 hr, washed with PBST twice or more, and incubated with about 1 ⁇ g/ml of the 3-4B and 47-235S monoclonal antibodies contained in 5% skim milk in PBST (PBS + 0.1% Tween 20) for 1 hr. After being washed with PBST five times, the blot was incubated with a 1:5000 dilution of anti-mouse Ig-HRP (Sigma) in 5% skim milk in PBST (PBS + 0.1% Tween 20), and was developed using ECL detection reagents (Amersham biosciences) .
• the 47-235S monoclonal antibody was found to detect a 47-kDa protein in Miz-hESl cells. However, the 47-235S monoclonal antibody did not bind to mouse-derived cells, STO, MEF and mESC(Jl), and to hNPSTl neural progenitor cells differentiated from human ES cells. These results indicate that the 47-235S monoclonal antibody recognizes only a human ES cell-specific protein, and that, as demonstrated in Example 6, it recognizes both a normal form of a protein antigen (conformational epitope) and a denatured form of the protein antigen (linear epitope) .
• the monoclonal antibodies of the present invention specifically recognize cell surface proteins of human ES cells.
• the monoclonal antibodies provide a tool for research into the difference between mice and humans as a higher animal species in the early embryonic development and thus are useful for the analysis of human ES cells.
• the monoclonal antibodies are useful for the removal of undifferentiated human ES cells for cell therapies.

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