KR100826597B1 - Monoclonal antibody specific to cell surface protein of human embroyonic stem cell - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically binds to a cell surface protein of human embryonic stem cells and a hybridoma that produces the same. More specifically, the present invention relates to a monoclonal antibody that specifically binds to cell surface proteins of human embryonic stem cells, A monoclonal antibody that does not bind to stem cells, a hybridoma that produces the monoclonal antibody, a black kit comprising the monoclonal antibody, and a composition for removing human embryonic stem cells.

Human embryonic stem cells, undifferentiated, cell surface proteins, monoclonal antibodies

Description

BACKGROUND ART [0002] Monoclonal antibodies specific for cell surface proteins of human embryonic stem cells are known as "             

1A shows the expression of human embryonic stem cell (1), alkaline phosphatase (2), negative control marker SSEA1 (3), positive control marker SSEA3 (4) and SSEA4 (5) A marker expression image showing the culture and characteristics of human embryonic stem cells,

FIG. 1B shows that OCT4 (octamer binding protein 4) gene, which is not expressed in mouse embryonic fibroblast (MEF), is expressed in human embryonic stem cells (hES) (Reverse Transcriptase-Polymerase Chain Reaction), and the product was observed by electrophoresis.

-RT: negative control without reverse transcriptase,

MEF: mouse embryonic fibroblast,

hES: human embryonic stem cells,

FIG. 1c shows a case in which a telomerase gene that is not expressed in MEF is expressed in a human embryonic stem cell, and a heat-sensitive telomerase gene is expressed in the human embryonic stem cell, and a telomere is additionally bound to increase telomere length by Southern blotting This is a photo

P: positive control extract and stepwise dilution,

P + heat: Positive control (sample) was tested for telomerase activity,

MEF: mouse embryo fibroblast extract,

hES: human embryonic stem cell extract,

hES + heat: heat treatment of human embryonic stem cell extract,

FIG. 2 is a graph showing that the monoclonal antibodies 3-4B and 47-235S of the present invention bind to human embryonic stem cells Miz-hES1 and HSF6 through fluorescent cell staining (the solid line is a monoclonal antibody, Is the second antibody only),

SSEA1: Antibody not binding to human embryonic stem cells (negative control),

SSEA3, 4: an antibody that binds to human embryonic stem cells (positive control),

3 is a photograph showing that the monoclonal antibodies 3-4B and 47-235S of the present invention bind to human embryonic stem cells, Miz-hES1 and Miz-hES4, through immunocyte staining.

SSEA1: Antibody not binding to human embryonic stem cells (negative control),

SSEA3, 4: antibody (positive control) binding to human embryonic stem cells,

4 is a graph showing that the monoclonal antibodies 3-4B and 47-235S of the present invention do not bind to mouse embryonic stem cells through fluorescent cell staining (the solid line is a monoclonal antibody and the red background is a secondary antibody only ),

SSEA4: Antibody not binding to mouse embryonic stem cells (negative control),

SSEA1: antibody binding to mouse embryonic stem cells (positive control),

FIG. 5 is a graph showing that the monoclonal antibodies 3-4B and 47-235S of the present invention do not bind to mouse embryonic fibroblasts through fluorescent cell staining (in this case, the solid line is a monoclonal antibody and the red background is a secondary antibody ),

FIG. 6 is a graph showing that the monoclonal antibodies 3-4B and 47-235S of the present invention do not bind to mouse fibroblast STO, which is a supporting cell used for culturing human embryonic stem cells, through fluorescent cell staining (the solid line in FIG. Monoclonal antibody and the red background contains only the secondary antibody).

7 is a graph showing that monoclonal antibodies 3-4B and 47-235S of the present invention do not bind to human neural precursor cells hNPST1 differentiated from human embryonic stem cells through fluorescent cell staining (the solid line represents a single clone And the red background contains only the secondary antibody).

FIG. 8 shows that the binding ability of the monoclonal antibodies 3-4B and 47-235S of the present invention to embryonic stem cells in the presence of retinoic acid inducing differentiation of human embryonic stem cells is decreased through fluorescent cell staining (At this time, the negative control group SSEA1, the positive control groups SSEA3 and SSEA4 were also included).

FIG. 9 is a figure showing the protein precipitated after immunotoluption with human monoclonal antibody 3-4B, 47-235S after biotinylation of human embryonic stem cells. The heavy chain below is a Western blotted photograph of the antibody used at this time (the same experiment was performed except for the antibody, which was used as a negative control).

FIG. 10 is a photograph showing Western blot analysis of the cell binding pattern of the monoclonal antibody 47-235S of the present invention (β-actin was subjected to western blotting in order to compare amounts of proteins used).

The present invention relates to a monoclonal antibody that specifically binds to the cell surface protein of human embryonic stem cells and a hybridoma producing the same.

A stem cell is a cell capable of differentiating the organisms that constitute a biological tissue into various cells, collectively referred to as undifferentiated cells before differentiation, which can be obtained from embryonic, fetal, and adult tissues . Stem cells are differentiated into specific cells by differentiation stimuli (environment), and unlike the differentiated cells in which the cell division is stopped, by self-renewal, the same cells as themselves are produced by cell division and proliferation ), And it is characterized by having plasticity in differentiation because it can be differentiated into other cells by different environment or differentiation stimulus.

Stem cells are divided into embryonic stem cells, which have pluripotency, and adult stem cells, which have multipotency. The inner cell mass of the blastocyte, which is an embryo in the early stage of embryogenesis, forms part of the embryo, and embryonic stem cells formed from the intracellular mass can theoretically be differentiated into cells of all the tissues constituting the individual It is a stem cell with potential. In other words, embryonic stem cells are undifferentiated cells capable of proliferating indefinitely. They can differentiate into all cells, and unlike adult stem cells, they can also produce germ cells, which can be inherited to the next generation. On the other hand, when the embryo development process proceeds and the embryos are introduced into the process of forming each organs of the fetus, the organs have specific stem cells (primary stem cells) And participate in the differentiation process. In this way, tissue-specific stem cells are generally limited to multipotent or unipotent cells that are capable of differentiating, and even after becoming adult, most of the organs have unique stem cells To compensate for the loss of normal or pathologically occurring cells.

In the historical background of embryonic stem cell research, Evans et al., Nature, 292: 151-156, 1981) established the first embryonic stem cell culture method in mice in 1981. In 1996, (Thomson et al., Biol. Reprod., 55: 254-259, 1996) was developed and human embryonic stem cell culture was established by Thomson et al. 282: 1145-1147, 1998). Therefore, since embryonic stem cells have differentiation ability to differentiate into all cells, it is expected that stem cells can be used to induce differentiation into specific cells in case of damage of specific cells or organs due to diseases or accidents, It is emerging as a fundamental treatment method for intractable diseases.

Although human embryonic stem cells have characteristics similar to those of the best known embryonic stem cells, there are also many distinctive parts. First, a representative similar feature is that human embryonic stem cells are cells that are capable of differentiating as if they are mice. Therefore, when embryoid bodies (EBs) are formed under in vitro conditions to induce differentiation, (Thomson et al., Science, 282: 1145-1147, 1998; Reubinoff, et al., Nat. Biotech., 18: 399-404; Park, et al., Biol. -2014, 2003). In the case of cell culture, both cells have similarity that they can be cultured in the presence of a feeder or feeder cell culture fluid. In addition, the Oct-4 gene, which is known to be involved in early differentiation in embryonic development, The presence of telomerase expressed in possible cells and the alkaline phosphatase highly expressed in mouse embryonic stem cells are commonly expressed in human embryonic stem cells. However, there are many differences between human and mouse embryonic stem cells. Although mouse embryonic stem cells can be cultured as a single cell, human embryonic stem cells can not be cultured as a single cell because most of them die when they are made into a single cell. Not only morphologically different, but also cytokine requirement to maintain self-renewal or pre-differentiation ability during cultivation is different. In the actual microarray results, we can see that the pool of genes to maintain the stameness of human embryonic stem cells is significantly different from that of mice (Bhattacharya, et al., Blood, 103: 2956-2964, 2004).

Nevertheless, in order to identify and define human embryonic stem cells in undifferentiated cultures currently being cultured, monoclonal antibodies prepared by injecting mouse embryo or human embryonal carcinoma, rather than human embryonic stem cells, For example, antibodies against TRA-1-60, TRA-1-81, SSEA1, SSEA3, and SSEA4 are used. However, most of the molecules recognized by these antibodies have carbohydrate epitopes and their function is not known yet (Badcock, et al., Cancer Res. 59: 4715-4719, 1999; Kannagi et al., EMBO. 2: 2355-2361, 1983). Although other protein markers, CD9, are also known to be expressed on human embryonic stem cell surfaces, they are also expressed in mouse embryonic stem cells (Oka, et al., Mol. Biol. Cell 13: 1274-1281, 2002; Carpenter, et al , Dev Dyn 229: 243-258, 2004). Therefore, in order to study undifferentiated human embryonic stem cells, it is urgently required to find more specific markers specific to human embryonic stem cells. In fact, human embryonic stem cells that are actually cultured in undifferentiated state are directly injected, Are expected to elicit cell surface molecules that are specific to a wide variety of human embryonic stem cells.

    Currently, the core of cell therapy using stem cells is the separation of stem cells from humans and animals, the establishment of stem cell culture technology, the induction and differentiation of specific functional cells into in vitro conditions, Establishment of safety, and suppression of immune rejection in human transplantation, among which the most important technology is the induction of differentiation into various functional cells. To date, mouse embryonic stem cell studies have shown that hematopoietic cells (Wiles et al., Development, 111: 259-267, 1991), cardiomyocytes (Klug et al., J. Clin. 98: 216-224, 1996), insulin-secreting cells (Soria et al., Diabetes, 49: 157-162, 2000), neurons and glia et al., Dev Biol., 168: 342-357, 1995; Okabe et al., Mech Dev., 59: 89-102,1996, Mujtaba et al., Dev Biol., 214: 1999, Brustle et al., Science, 285: 754-756, 1999; Brustle et al., Proc. Natl. Acad. Sci. USA, 94: 14809-14814, 1997) Cell-based studies are in the process of inducing and establishing the differentiation into specific functional cells by applying the already established method of culturing mouse embryonic stem cells. (Kehat, et al., Circ. Res 91: 659-661, 2002; Mummery et al., ≪ RTI ID = 0.0 > 19: 1134 - 1140, 2001), neural precursors (Zhang et al., Nat Biotechnol., 19: 1129-1133, Reubinoff et al., Nat Biotechnol., 19: The possibility of differentiation into various cells such as vascular endothelial cells (Levenberg, et al., PNAS. 99: 4391-4396, 2002), hematopoietic cells (Chadwick et al., Blood, 102: 906-915, 2003) In the future, it is expected that the induction of differentiation into various cells will be reported.

As described above, when various functional cells induced to differentiate from human embryonic stem cells are used for the cell therapy, it is important that the efficacy and safety be established in an actual animal or human body by first separating them. Although human embryonic stem cells themselves can be used directly in cell therapy for a variety of degenerative diseases, this case is known to cause cancer in mice and the like (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). Therefore, functional cells induced to differentiate from human embryonic stem cells for cell therapy must be used for cell therapy after completely eliminating human embryonic stem cells. However, antibodies that identify currently used undifferentiated human embryonic stem cells are difficult to analyze their characteristics more accurately and to separate these undifferentiated cells. Therefore, development of more antibodies that specifically recognize human embryonic stem cells can be useful for accurately analyzing the characteristics of human embryonic stem cells and for removing human embryonic stem cells during cell therapy.

Under these circumstances, the present inventors have found that human embryonic stem cells cultured in human embryonic stem cells are undifferentiated human embryonic stem cells, and then used in the production of monoclonal antibodies specific for human embryonic stem cells The present inventors have completed the present invention by confirming that a monoclonal antibody binds specifically to human embryonic stem cell surface proteins and is a monoclonal antibody that does not bind to mouse embryonic stem cells.

It is an object of the present invention to provide a monoclonal antibody that specifically binds to the cell surface protein of human embryonic stem cells and does not bind to mouse embryonic stem cells.

It is another object of the present invention to provide a hybridoma producing the above monoclonal antibody.

Another object of the present invention is to provide a test kit for undifferentiated human embryonic stem cells comprising the above monoclonal antibody.

It is still another object of the present invention to provide a composition for removing undifferentiated human embryonic stem cells comprising the above monoclonal antibody.

It is still another object of the present invention to provide a method for removing undifferentiated human embryonic stem cells using the above monoclonal antibody.

In one embodiment, the present invention relates to a monoclonal antibody that specifically binds to cell surface proteins of human embryonic stem cells and does not bind to mouse embryonic stem cells.

In one specific embodiment, the monoclonal antibody of the invention binds to undifferentiated human embryonic stem cells and does not bind to undifferentiated human embryonic stem cells.

In another specific embodiment, the monoclonal antibody of the present invention specifically binds to a human embryonic stem cell protein having a molecular weight of about 26 kDa on 10% SDS-PAGE.

In another specific embodiment, the monoclonal antibody of the present invention specifically binds to human embryonic stem cell protein having a molecular mass of about 47 kDa on 10% SDS-PAGE.

In a preferred embodiment, the monoclonal antibody of the invention is monoclonal antibody 3-4B produced by hybridomas of accession number KCTC 10599BP.

In another preferred embodiment, the monoclonal antibody of the invention is monoclonal antibody 47-235S produced by hybridoma of accession number KCTC 10739BP.

As used herein, the term " monoclonal antibody " refers to a protein molecule that is directed against and specifically binds to a single antigenic site (single epitope). For the purpose of the present invention, the monoclonal antibody of the present invention specifically binds to the cell surface proteins of undifferentiated human embryonic stem cells, and thus is a protein molecule that recognizes cell surface proteins of undifferentiated human embryonic stem cells.

Since the variable regions of heavy and light chains, particularly the complementarity determining regions (CDRs), contribute to the formation of such complexes, the major sites of antibodies that recognize specific epitopes of the antigen and form an antigen-antibody complex, A chimeric antibody, a humanized antibody, etc., including a CDR, are included in the scope of the present invention. The present invention also includes functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains as long as they have the binding properties as described above. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv.

In order to produce a monoclonal antibody specific for undifferentiated human embryonic stem cells, the present inventors have succeeded in culturing human embryonic stem cells in a large amount with ease by using a collagenase enzyme for subsequent culture, analyzing the characteristics thereof, After embryonic stem cells were identified, they were used to immunize mice.

Specifically, after human embryonic stem cells were cultured, the morphology of human embryonic stem cells and the expression of alkaline phosphatase were observed with a phase contrast microscope (see 1 in FIG. 1A and 2 in FIG. 1A) by hematoxylin and eosin staining, (Fig. 1C) and RT4-PCR (Fig. 1B), indicating that the cultured cells were human embryonic stem cells. In addition, through SSEA (stage specific embryonic antigen) staining, the antibody against SSEA1, which is a negative marker for human embryonic stem cells, does not bind to the cells but binds to antibodies against SSEA3 and SSEA4, which are positive markers, (See Figs. 3, 4 and 5 of Fig. 1a). As a result, it has become possible to produce a monoclonal antibody that specifically binds to undifferentiated human embryonic stem cells using the embryonic stem cell itself as an antigen.

Then, the cultured human embryonic stem cells are inactivated, and then the mouse is immunized and the splenocytes isolated therefrom are fused with cancer cells. From the hybridomas, monoclonal antibody 3- 4B and 47-235S were isolated and purified to confirm the binding ability of the antibody to human embryonic stem cells (see FIG. 2). The mouse embryonic stem cells, mouse embryonic fibroblasts, and mouse fibroblasts (STO) (Figs. 4 to 6). It was also confirmed that these antibodies decreased binding ability in neuronal precursor cells hNPST1 (Korean Patent Application No. 10-2004-0011705) and cells differentiated by treatment with retinoic acid, which are differentiated from human embryonic stem cells (Figs. 7 and 8). In addition, 10% SDS-PAGE showed that the monoclonal antibody 3-4B had a molecular weight of about 26 kDa and the monoclonal antibody 47-235S recognized a protein of human embryonic stem cells having a molecular weight of about 47 kDa (FIG. 9) .

The molecular weight of the protein of human embryonic stem cells recognized by the monoclonal antibody of the present invention is measured using 10% SDS-PAGE and may be varied within a certain range depending on the measurement conditions of the molecular weight. Thus, the use of the term " about " is unavoidable in presenting the molecular weight of the protein, and can generally range from +/- 2 kDa, preferably +/- 1 kDa.

Unlike the present invention in which monoclonal antibodies were prepared using human embryonic stem cells, human embryonic stem cell-recognizing antibodies were prepared using mouse embryos or human embryonic carcinoma cells before the present invention. Examples of such antibodies are SSEA3, SSEA4, TRA-1-60, 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). Although these antibodies bind to human embryonic stem cells, the biological function of these antibodies is unclear because they contain carbohydrates, not proteins. Therefore, the monoclonal antibody of the present invention which binds to the protein on the cell surface of human embryonic stem cells differs from the antibody that has been developed before the present invention as described above.

On the other hand, CD9 is known as an antibody recognizing the cell surface protein of human embryonic stem cells, but this antibody is an antibody that binds not only human embryonic stem cells but also mouse embryonic stem cells (Oka, et al., Mol. Biol Cell 13: 1274-1281, 2002; Carpenter, et al., Dev Dyn 229: 243-258, 2004). This characteristic is clearly distinguished from the monoclonal antibody of the present invention which binds specifically to human embryonic stem cells without binding to mouse embryonic stem cells. The antibody against CD9 is expressed in both human and mouse embryonic stem cells While the monoclonal antibody of the present invention recognizes an antigen protein existing only in human embryonic stem cells.

Thus, antibodies to embryonic stem cells can selectively bind to embryonic stem cells such as humans and mice in other species. For example, mouse embryonic stem cells are associated with antibodies to SSEA1, but SSEA3 Human embryonic stem cells do not bind to the antibody to SSEA1 but to SSEA3 and to SSEA4 (Thomson et al., Science, 282: 1145-1147, 1998). In addition, antibodies to TRA-1-60 and TRA-1-81 derived from human embryonic carcinoma cells do not bind to mouse embryonic stem cells but bind to human embryonic stem cells (Andrews, et al., Hybridoma 3: 347- 361, 1984).

Therefore, the binding specificity of the monoclonal antibody of the present invention, which specifically binds to the cell surface protein of human embryonic stem cells but does not bind to mouse embryonic stem cells, is determined by the binding specificity of the monoclonal antibody of the present invention, Lt; RTI ID = 0.0 > monoclonal < / RTI >

In particular, since the monoclonal antibody 47-235S of the present invention recognizes not only the normal conformational protein epitope but also the denatured protein epitope, it is more preferable to analyze the biological function of human embryonic stem cells Lt; / RTI >

In another aspect, the present invention relates to a hybridoma producing the monoclonal antibody of the present invention as described above.

In one specific embodiment, the invention provides a hybridoma producing the monoclonal antibody 3-4B or 47-235S.

In a specific embodiment, the hybridoma of the present invention irradiates human embryonic stem cells with radiation to inactivate the cells; Injecting the inactivated embryonic stem cells into the abdominal cavity of a mouse; Isolating lymphocytes from the spleen of said mice; And the lymphocytes were fused with myeloma cancer cells.

Hybridomas secreting monoclonal antibody 3-4B were designated as hybridoma 3-4B (Accession No: KCTC 10599BP), respectively, and were named KCTC (Korean Collection for Type Cultures, Korea Korea Research Institute of Bioscience & Biotechnology 52, Eun-dong, Yuseong-gu, Daejeon, Korea). The hybridoma that secretes the above monoclonal antibody 47-235S was designated as Hybridoma 47-235S (Accession No .: KCTC 10739BP), which was deposited with KCTC on December 6, 2004.

Hybridomas that secrete monoclonal antibodies can be cultured in vitro or in vivo in vitro.

The monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma may be purified to a high purity (for example, 95% or more) according to a method well known in the technical field of the present invention .

Such purification techniques can be separated from the culture medium or ascites fluid using, for example, purification methods such as gel electrophoresis, dialysis, salt precipitation, chromatography, and the like.

In a specific embodiment of the present invention, for the mass production of monoclonal antibodies of the present invention, hybridomas were injected into the abdominal cavity of mice and cultured in duplicate and separated using protein G-sepharose column chromatography.

In another aspect, the present invention relates to a composition for removing undifferentiated human embryonic stem cells comprising the above monoclonal antibody.

In another aspect, the present invention relates to a method for removing undifferentiated human embryonic stem cells using the above monoclonal antibody.

The monoclonal antibody of the present invention can be used to remove embryonic stem cells present in cells to be transplanted for cell therapy or to remove embryonic stem cells present in transplanted cells.

In order to selectively remove embryonic stem cells, the monoclonal antibody of the present invention can be indirectly coupled (for example, covalently bonded) to a known therapeutic agent directly or through a linker or the like. Therapeutic agents that may be conjugated to the antibody include, but are not limited to, radionuclides, drugs, lymphokines, toxins, and antibodies capable of differentiating.

To remove embryonic stem cells present in the transplanted cells, the antibody may be administered as such or in a composition comprising the antibody. In the case of a composition comprising an antibody, an appropriate preparation is prepared containing an acceptable carrier depending on the mode of administration. Formulations suitable for the mode of administration are well known in the art. These agents are administered by any suitable method including, but not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if necessary, for localized immunosuppressive therapy. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. The composition comprising the antibody of the present invention may be administered in a pharmaceutically effective amount to remove embryonic stem cells. Typical dosage levels can be optimized using standard clinical techniques.

In another aspect, the present invention relates to a test kit for undifferentiated human embryonic stem cells comprising the above monoclonal antibody.

The monoclonal antibodies of the present invention can be used not only for the removal of embryonic stem cells in transplanted or transplanted cells through an antigen-antibody complex reaction, but also for specifically detecting undifferentiated human embryonic stem cells.

These assay kits include monoclonal antibodies of the present invention as well as tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

Antigen-antibody complex formation can be induced by immunofluorescent staining, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, (Complement Fixation Assay), FACS, protein chip, and the like.

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes . Enzymes that can be used as detection labels include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, Oxydase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, β - Latamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3] 2+ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radiation isotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1 Culture of Human Embryonic Stem Cells and Characterization of Embryonic Stem Cells

<1-1> Culture of human embryonic stem cells

In order to produce a novel monoclonal antibody capable of specifically recognizing human embryonic stem cells, the inventors of the present invention developed a human embryonic stem cell Miz-hES1 (Park, et al., Biol. Reprod. 69: 2007-2014, 2003 ), Miz-hES4 (Kim, et al Mole & Cells 2004 In press), HSF6 (Abeyta, et al., Human Mol. Genet 13: 601-608, 2004) (Dibbecco's modified Eagle`s medium) / F12 (Gibco, Rockville, MD, USA), 20% knockout SR (Gibco), 0.1 mM mercaptoethanol (Sigma, St Louis, MO, USA), 2 mM glutamine (Gibco), 0.1 mM nonessential amino acid (Gibco) Human embryonic stem cells were cultured in a medium of streptomycin (Sigma) and 4 ng / ml of bFGF (Gibco Invitrogen), followed by subculture at 6-day intervals.

Specifically, a 12-well tissue culture plate (Nunclon) was coated with a 0.1% gelatin solution at 37 ° C for 10 minutes, and then MEF (mouse embryonic fibroblast, Korea Experimental Animal Room, Biotechnology Research Institute) was inoculated at 6.5 × 10 ⁴ cells / well. The irradiated MEF does not grow but is a cell that supports the growth of human embryonic stem cells. Human embryonic stem cell tissue, which had been grown for 5-7 days after 24 hours of MEF culture, was treated with 1 mg / ml of collagenase IV (Gibco) for 1 hour at 37 ° C, and then the stem cells were cut to an appropriate size Then, the cells were transferred to a plate for MEF tissue culture prepared as described above, and cultured for 48 hours.

<1-2> Observation of Human Embryonic Stem Cells by Hematoxylin and Eosine Staining

Human embryonic stem cells cultured for 6-7 days cultured by the method of Example <1-1> were washed with PBS (phosphate buffered saline), and then treated with 4% paraformaldehyde (Roche , NY, USA) for 30 min. The cells were washed three times with PBS, and treated with 100%, 95%, 80%, and 70% ethanol for 1 min each. The cells were then washed with flowing water and then treated with hematocylin (Sigma) for 5 minutes. After 5 minutes, the hematocylline was removed with ammonia water and stained with 0.5% eosin (Eosin, Sigma) for 5 minutes to stain the cytoplasm of stem cells. Finally, the cells were washed again with ammonia water and treated with 70%, 80%, 95%, and 100% ethanol for 1 minute each, and then observed by phase contrast microscopy (FIG. As a result, the cells formed a clear boundary with the MEF cells and all the cells were closely connected to each other to form a flat globular lump, which confirmed the characteristic morphology of human embryonic stem cells.

<1-3> Expression of alkaline phosphatase (Alkaline phosphatase)

Human embryonic stem cells cultured by the method of Example <1-1> were fixed with 4% paraformaldehyde for 30 minutes and then washed again with PBS. Then, 2% tween 20 was treated for 30 minutes, washed with distilled water, and stained for 15 minutes using an AP staining kit (Sigma) according to the manufacturer's manual. Finally, after washing with distilled water, hematoxylin was treated for 2 minutes by counterstaining and observed under a phase contrast microscope (Fig. 1a, 2). As a result, it was confirmed that the blue stain was stained with hematoxylin, and the alkaline phosphatase enzyme was expressed and the red cell mass was stained.

<1-4> SSEA staining

Human embryonic stem cells cultured by the method of Example <1-1> were fixed with 4% paraformaldehyde for 30 minutes, washed again with PBS, and then blocked with normal horse serum for 1 hour. Subsequently, antibodies against SSEA (stage specific embryonic antigen) 1, SSEA3, and SSEA4 (DSHB, the University of Iowa, USA) were treated for 1 hour and washed with PBS. Finally, the secondary antibody against SSEA was treated for 1 hour, treated with Vectastain ABC reagent (DAP staining kit, Sigma) for 20 minutes, washed with PBS, treated with substrate solution and stained, and immunohistochemically As a result, human embryonic stem cells were found to be negative for SSEA1, which is a negative control marker, and positive for SSEA3 and SSEA4, which are positive control markers (3, 4 and 5 in FIG. 1a).

<1-5> Measurement of telomerase activity

The fact that the telomerase gene, which is not expressed in MEF (mouse embryonic fibroblast), is expressed in human embryonic stem cells, and that the heat-sensitive telomerase gene is expressed and has an increased length of telomeres by further binding of telomere blotting).

Specifically, the human embryonic stem cells cultured by the method of Example <1-1> were treated with collagenase and separated into individual cells. Then, telomerase activity assay kit (Intergen, NY , USA) was used to perform PCR after cell lysis according to the manufacturer's manual. According to the manufacturer's instructions, telomerase was added to the TS primer to extend the telomeres. The RP primer and Taq polymerase were used for 30 seconds at 94 ° C, 30 seconds at 59 ° C, 36 minutes at 72 ° C for 1 minute PCR was performed. After the PCR was performed, the product was electrophoresed on a 2% agarose gel, and the DNA was transferred to the membrane. Subsequently, the DNA was transferred to a membrane using a probe of the nucleotide sequence of SEQ ID NO: 5 labeled with ³² P radioactive isotope, Southern blotting) to measure telomerase activity. As a result, it was confirmed that the human embryonic stem cells express heat-sensitive telomerase (FIG. 1C). In FIG. 1C, P is a positive control and its diluent, P + heat is a positive control (sample) inactivation of telomerase activity by heat treatment, MEF is mouse embryonic fibroblast, hES is human embryonic stem cell And hES + heat represents the heat treatment of the human embryonic stem cell extract.

<1-6> Oct-4 Expression Survey

Total RNA was isolated from human embryonic stem cells cultured by the method of Example <1-1> using an RNA isolation kit (Roche), and the Oct4 gene, which is not expressed in mouse embryonic fibroblasts, In order to confirm expression in human embryonic stem cells, RT-PCR using the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 specific to the Oct4 gene, and SEQ ID NO: 3 and SEQ ID NO: 4 for RNA quantification, RT-PCR was performed using an actin primer. After performing the PCR, the product was electrophoresed on an agarose gel of 1.5% to confirm the expression of Oct4 gene (FIG. 1B). In FIG. 1B, -RT is a negative control without reverse transcriptase, MEF is mouse embryonic fibroblast, and hES is human embryonic stem cell.

Example 2 Preparation of Mouse Hybridoma

<2-1> Immunization of human embryonic stem cells into mice

The human embryonic stem cell Miz-hES1 cultured by the method of Example <1-1> was treated with collagenase IV and separated, and about 2 × 10 ⁶ cells were suspended in 100 μl of PBS and γ-radiation The stem cells were inactivated and injected into the abdominal cavity of a Balb / c mouse (laboratory animal laboratory, Korea Research Institute of Bioscience and Biotechnology). Three times at 3-week intervals, and then injected again 3 days before cell fusion.

<2-2> Preparation of mouse hybridoma producing monoclonal antibody

To collect feeder cells, 20 ml of DMEM (GIBC0) medium was added to the peritoneum of healthy mice the day before cell fusion, and the cells in the abdominal cavity were collected by centrifugation, The cells were extracted and mixed. The cells were mixed with 20% fetal bovine serum, and the cells were divided into 96-well plates so as to have 10 ⁵ cells per well. The cells were cultured in a CO ₂ incubator at 37 ° C. NS1 myeloma cell line (ATCC, USA) to be fused with spleen cells from 2 weeks ago was also cultured in a mixture of RPMI1640 (GIBCO) and 10% fetal bovine serum.

As in Example <2-1>, the spleen was taken out from the mice immunized with human embryonic stem cells, washed well with RPMI1640 (GIBCO), finely pulverized in a Petri dish with a glass rod, and the cell suspension was left in a 15 ml tube, The supernatant was transferred to a new tube when it was submerged. NS1 was harvested by centrifugation and suspended in 10 ml of RPMI1640 to count the cells together with the splenocytes. 10 ^Seven NS1 and 10 ⁸ spleen cells were transferred to a 50 ml tube and centrifuged at 200 xg for 5 min to remove supernatant and placed in a 37 ° C water-filled beaker for 2 min. 1 ml of PEG solution (GIBCO) was added for 1 minute while gently shaking the tube by gently tapping the tube and shaking gently with immersion in water at 37 ° C. After centrifugation at 100 x g for 2 minutes, 5 ml of RPMI1640 solution was slowly added over 3 minutes, and 5 ml of RPMI1640 solution was slowly added over 2 minutes. Then, the cells were recovered by centrifugation at 200 x g, (RPMI 1640 + 20% fetal bovine serum). The cells were allowed to stand for 30 minutes in a CO ₂ incubator at 37 ° C, and then they were divided into 10 ⁵ cells in a 96-well plate containing MEF cells (feeder cells) preliminarily cultured in a CO ₂ incubator at 37 ° C in a CO ₂ incubator Lt; / RTI &gt; The next day, 70 [mu] l HAT was added and colonies grew growing in HAT medium for 3 days at 2 weeks.

A sandwich ELISA (Enzyme Linked Immunosorbent Assay) method was used to select clones expressing the antibody. 100 μl of the hybridoma culture was added to a plate coated with anti-mouse IgG or IgM antibody at 2 μg / ml, followed by reaction at 37 ° C for 1 hour. HRP (horseradish peroxidase, Sigma) For 1 hour. The plate was washed with phosphate buffer solution supplemented with 0.05% Tween 20. OPD (Sigma) and a substrate solution containing H ₂ O ₂ were added, and the absorbance at 492 nm was measured to select the clone producing the antibody Respectively.

Example 3 Production of Monoclonal Antibody Binding to Human Embryonic Stem Cells

<3-1> Selection of a hybridoma clone producing monoclonal antibody binding to human embryonic stem cells

Among the clones prepared by the method of Example 2, the ability of the hybridoma supernatant to secrete antibodies relatively stably to human embryonic stem cells was examined. Specifically, the cultured human embryonic stem cells were separated by collagenase IV and treated with GIBCO for 20 minutes at 37 ° C to separate into single cells, which were then passed through a 40 μm strainer to obtain 2 × 10 ⁵ cells were used for flow cytometry. First, single-celled human embryonic stem cells were suspended in PBA (1% BSA dissolved in PBS) and the antibody supernatant was reacted at 4 ° C for 30 minutes. 100 μl of the supernatant was removed by centrifugation at 1200 rpm for 5 minutes at 4 ° C., followed by reaction with anti-mouse Ig-FITC (BD) at 200 ° C. for 30 minutes at 4 ° C., followed by washing twice with PBA, Only the cells negative to Propidium Iodide (PI) were selected and analyzed for binding capacity to human embryonic stem cells using FACS caliber (caliber).

As a result, a variety of hybridomas secreting antibodies binding to human embryonic stem cells were selected and subcloning continued with subculture to obtain antibodies 3-4B which retained their stability with certainty and maintained their specificity for human embryonic stem cells , A hybridoma secreting 47-235S antibody was selected.

The hybridoma secreting the monoclonal antibody 3-4B was designated as Hybridoma 3-4B (Accession No: KCTC 10599BP), which was deposited with KCTC (Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology).

The hybridoma that secretes the above monoclonal antibody 47-235S was named Hybridoma 47-235 (Accession No .: KCTC 10739BP) and deposited with KCTC on December 6, 2004.

<3-2> Purification of Monoclonal Antibody

Monoclonal antibodies 3-4B and 47-235S were isolated from the hybridomas 3-4B and 47-235S selected in the above Example <3-1>, respectively.

Specifically, to purify the antibody 3-4B, 47-235S, the hybridoma cell 1 × 10 ⁷ was dissolved in 0.5 ml of PBS in peritoneal cavity of 0.5 ml of pristane-inoculated Balb / c mice one week before After 10-14 days of injection, the extracts were extracted with a syringe, and the ascites was centrifuged to collect only the supernatant. PBS was added to 1 mL of the multiple solution, and the solution was diluted to 2 mL. Further, 1 nM of EDTA and 0.02% of NaN ₃ were added to the plural liquids, and they were filtered with a 0.22 탆 filter. The antibody was bound using a protein G-Sepharose column (Pharmacia, Sweden) for 2 hours at 4 ° C, the column was placed upright and the wall of the column was washed with wash buffer (0.5 M NaCl, 0.1 M Tris, pH 8.0) and the column was connected to a peristatic pump and thoroughly washed with a washing solution. After completion of washing, the antibody was eluted with 0.2 M glycin-HCl (pH 2.7). At this time, the eluate was buffered in a tube containing 1 M of Tris (pH 9.0) prepared in advance. In the following, the 3-4B, 47-235S antibody was selected and the next step was started.

Example 4: Analysis of specificity of monoclonal antibody

The binding ability of 3-4B and 47-235S antibodies purified in Example <3-2> to human embryonic stem cells was examined by fluorescent cell staining in the same manner as in Example <3-1> (Fig. 2 ). SSEA1 is an antibody (negative control) that does not bind to human embryonic stem cells. SSEA4 is an antibody that binds to human embryonic stem cells (positive control) )to be.

Human embryonic stem cells Miz-hES1 and Miz-hES4 were fixed with 4% paraformaldehyde for 30 minutes in the same manner as in the above Example <1-4>, and the cells were treated with SSEA1, SSEA4, 3-4B, and 47-235S Were observed by immunocytochemistry. SSEA1 was negative and SSEA3 and SSEA4 were positive for the negative control marker SSEA1 and the positive control marker SSEA3 and SSEA4 (FIG. 3). Both monoclonal antibodies 3-4B and 47-235S were found to bind well to both Miz-hES1 and Miz-hES4 (Fig. 3).

In addition, mouse embryonic fibroblasts (J1) (Li. Et al., Cell, 69: And 10% fetal bovine serum, and then were separated into collagenase IV. In order to confirm the binding ability of the 3-4B and 47-235S antibodies to mouse embryonic stem cells, mouse embryonic fibroblasts, and mouse fibroblast STO cells by the same method as above, flow cytometry was performed by fluorescent cell staining (Fig. 4, Fig. 5, and Fig. 6). As a result, it was confirmed that 3-4B, 47-235S antibody did not bind to the cell line. And also did not bind to human neural precursor cells (hNPST1) induced differentiation in human embryonic stem cells (Fig. 7).

Example 5 Monoclonal Antibody Analysis on Differentiated Human Embryonic Stem Cells

Since human embryonic stem cells have the property of losing undifferentiated ability and differentiating by treatment with retinoic acid (Henderson, et al., Stem Cells 20: 329-337, 2002), 10 days in Miz-hES1 medium cultured on day 4 ^-5 M retinoic acid was or was not treated for 6 days, and then the cells were removed and subjected to FACS analysis as in Example <3-1> as an antibody (FIG. 8). As a result, it was confirmed that the binding capacity was decreased in the differentiated cells, and it was found that this antibody specifically binds to undifferentiated human embryonic stem cells.

<Example 6> Analysis of antigens recognized by monoclonal antibodies by immunoprecipitation

In order to isolate the cell surface markers of human embryonic stem cells recognized by the monoclonal antibodies 3-4B and 47-235S, the human embryonic stem cells that had been cultured first were washed with PBS and stained with EZ-Link Sulfo-NHS-LC-Biotin (Pierce, The cells were incubated in a solution (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 / / ml leupeptin) for 20 minutes at 4 째 C and nuclei were removed by centrifugation. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). Proteins that bind nonspecifically to Protein G plus-Sepharose (Santa Cruz Biotechnology; Santa Cruz) were incubated with 20 μl of Protein G plus-sepharose for 2 hours at 4 ° C and centrifuged to recover supernatant After removing the supernatant, the supernatant was again reacted with about 1 μg of antibody at 4 ° C for 12 hours. Then, 20 μl of Protein G plus-sepharose was added thereto, followed by reaction at 4 ° C for 2 hours. Respectively. The recovered precipitate was washed with the cell lysate more than 10 times and the remaining protein was separated on 10% SDS-PAGE. This protein was Western blotted on a nitrocellulose membrane. The nitrocellulose membrane was reacted with PBST (PBS + 0.1% Tween 20) containing 5% skim milk for 1 hour, washed twice with PBST, and then streptavidin-HRP (horseradish peroxidase) conjugate (1: 1,500 Amershambiosciences) And reacted for 1 hour. After 5 washes with PBST, bio-labeled proteins were developed with ECL detection reagent (Amersham biosciences) (Fig. 9). As a result, it was confirmed that the monoclonal antibody 3-4B binds to a protein having a molecular weight of about 26 kDa. Monoclonal antibody 47-235S antibody binds to a protein of about 47 kDa in size.

Example 7 Analysis of 47-235S Antibody by Western Blot

10% SDS-PAGE was performed using a cell lysate of STO, MEF, MESC (J1), Miz-hES1, and hNPST1 cells to examine whether the antibody produced in the above example could recognize the antigen even in the denatured state Respectively. Western blotting was performed on the nitrocellulose membrane as in Example 6 above. The nitrocellulose membrane was reacted with PBST (PBS + 0.1% Tween 20) containing 5% skim milk for 1 hour and then washed twice more with PBST. The monoclonal antibody 3-4B, 47-235S was then added to about 1 μg / ml in PBST (PBS + 0.1% Tween 20) containing 5% skim milk for 1 hour. After 5 washes with PBST, anti-mouse Ig-HRP (Sigma) was diluted in PBST (PBS + 0.1% Tween 20) containing 5% skim milk at 1: 5000 and the labeled proteins were analyzed with ECL detection reagent (Amersham biosciences). As a result, monoclonal antibody 47-235S was found to detect 47kDa protein in Miz-hES1. However, it did not bind to mouse derived cells, MEF, STO, mESC (J1) and hNPST1, a neural precursor cell differentiated from human embryonic stem cells. These results indicate that monoclonal antibody 47-235S recognizes only proteins specific to embryonic stem cells and recognizes normal conformational epitopes as shown in Example 6 as well as epitopes of linear epitopes ) &Lt; / RTI &gt;

As described above, since the monoclonal antibody of the present invention specifically recognizes the cell surface protein of human embryonic stem cells, it is possible to provide a tool for studying differences in initial embryonic development of mice and humans, It can be used for the analysis of embryonic stem cells and can be usefully used to remove undifferentiated human embryonic stem cells in cell therapy.

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Claims (10)

A monoclonal antibody that specifically binds to the cell surface protein of human embryonic stem cells, produced by a hybridoma with accession number KCTC 10599BP. A monoclonal antibody that specifically binds to the cell surface protein of human embryonic stem cells, produced by a hybridoma with accession number KCTC 10739BP. delete delete delete delete Hybridoma with accession number KCTC 10599BP. Hybridoma with accession number KCTC 10739BP. A composition for the removal of undifferentiated human embryonic stem cells, comprising the monoclonal antibody of claim 1. 6. A kit for assaying undifferentiated human embryonic stem cells, comprising the monoclonal antibody of claim 1.

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