WO2020085563A1 - Monoclonal antibody n16-f2 specific to human naive pluripotent stem cells - Google Patents

Abstract

The present invention relates to: a monoclonal antibody specifically binding to human naive pluripotent stem cells; and a hybridoma producing same and, particularly to: monoclonal antibody N16-F2, which binds to human naive pluripotent stem cells but not to human primed pluripotent stem cells or has significantly weak binding capacity; hybridoma N16-F2 for producing the monoclonal antibody; an assay kit comprising monoclonal antibody N16-F2; and a composition for removing human pluripotent stem cells.

Description

Monoclonal antibody N16-F2 specific for human pure pluripotent stem cells

The present invention relates to a monoclonal antibody N16-F2 specifically binding to human naive pluripotent stem cells and a hybridoma producing the same.

Pluripotent stem cells are cells that have the ability to differentiate into all the cells that make up the body and were first isolated from the blastocysts of early embryos of mice, monkeys and humans (Evans et al., 1981, Nature, 292: 151; Thomson et al., 1995, PNAS 92: 7844; Thomson et al., 1998, Science, 282: 1145). The first human pluripotent stem cell, human embryonic stem cell (hESC), was established in human frozen embryos by Dr. James Thomson, USA in 1998 (Thomson et al, 1998, Science, 282: 1145), 2007 In 2011, the human induced Pluripotent Stem Cell (iPSC), similar to human embryonic stem cells, was established by gene introduction by Dr. Shinya Yamanaka of Japan (Takahashi et al., 2007, Cell 131: 861), In 2013, a human cloned embryonic stem cell was produced by nuclear transfer of an egg by Dr. Shoukhrat Mitalipov of the University of Oregon, USA (Tachibana et al., 2013, Cell 153: 1228), and currently there are three human pluripotent stem cells. . Pluripotent stem cells are characterized by self-renewal, which can infinitely produce the same cells as themselves by cell division, and pluripotency, which can differentiate into all functional cells in the body by various environments and differentiation stimuli. It is expected that it can be used to induce differentiation into specific cells in case of damage to certain organs or cells due to disease or accident. Therefore, human pluripotent stem cells capable of differentiating into all human cells are emerging as key stem cells for future regenerative medicine (Hackett and Surani 2014, Cell Stem Cell 15: 416). In fact, in a recent study, University College London and Moorfields Ophthalmology Hospital in the UK have already successfully demonstrated the possibility of recovering vision using retinal pigment epithelial cells derived from human embryonic stem cells in elderly geriatric degeneration patients, the number 1 cause of blindness in the elderly ( Cruz et al., 2018 Nature Biotechnology 36: 328).

In 2007, different types of pluripotent stem cells from embryonic stem cells and induced pluripotent stem cells were simultaneously isolated from mouse epidermal cells by British and American researchers and named epiblast-derived stem cells (EpiSC). Like the mouse embryonic stem cells, it has the pluripotency and can form a trilobite, but there are many differences in the cell morphology and molecular characteristics of the mouse embryonic stem cells, but rather the morphological and molecular characteristics of the human embryonic stem cells. Showed similar characteristics (Tesar et al., 2007 Nature 448: 196; Brons, et al 2007 Nature, 448: 191). Therefore, Dr. Austin Smith of Cambridge University said that mammalian pluripotent stem cells exist in two different states, and the embryos are divided into before and after implantation in the uterus, resulting in pre-implantation epiblast or post-implantation epiblast. Each of the pluripotent stem cells can be separated, and the pluripotent stem cells before implantation were named as naive or ground pluripotency, and after implantation as primed pluripotency (Nichols & Smith, 2009. Cell Stem Cell) 4: 487). Pure and quasi-state pluripotent stem cells have many differences in cell shape, growth rate, differentiation ability, degree of methylation, and germline transmission ability.The quasi-state grows in a large flat state and has a slow growth rate, but the pure state It grows in a small dome shape, grows well even when detached as a single cell with trypsin, grows fast, is expected to differentiate into more cells in a state that is not yet biased in the development stage, has a lower degree of methylation, and transmits the gonads. It is known to be an early pluripotent stem cell with superior ability (Hackett and Surani 2014, Cell Stem Cell 15: 416). Human embryonic stem cells, like pure mouse embryonic stem cells, are derived from pre-implantation blastocysts, but are very similar to epithelial stem cells (EpiSC) isolated from the upper germ cells after implantation of the mouse and can be classified as quasi-pluripotent stem cells. It can be seen that by applying the semi-embryonic stem cell cultivation method, it was cultured in a quasi-pluripotent state established by returning to a quasi-state like mouse EpiSC (Tesar et al., 2007 Nature 448: 196; Davidson et al., 2015, Development 142: 3090 ).

Meanwhile, other human pluripotent stem cells, i.e., pluripotent stem cells, cloned embryonic stem cells, etc., all exhibit quasi-derived post-implantation characteristics, and therefore, studies to establish pure pluripotent stem cells, i.e., pluripotent stem cells, before implantation in the human embryonic stage Has attracted high attention. When culturing mouse embryonic stem cells, two chemical inhibitors, mitogen activated protein kinase (MEK) inhibitors and glycogen synthase kinase 3 (GSK3) inhibitors (2i), can be used to suppress differentiation signals. ) Was added to the medium to maintain a high purity (Ying et al., 2008, Nature 453: 519), and in 2010, MIT's Rudolf Jaenisch group of Klf4, Klf2, The Oct4 gene was introduced and expressed, and pure human pluripotent stem cells were derived from metastable stem cells in LIF and 2i medium, and pure human iPSCs were also established through reprogramming of human somatic cells under the same culture conditions (Hanna et. al., 2010, PNAS 107: 9222). The University of Cambridge Austin Smith team introduced the Nanog, Klf2 gene, and then induced it into human pure pluripotent stem cells by a culture method comprising LIF, 2i, PKC inhibitor, and ascorbic acid (Takashima et al., 2014, Cell 158: 1254). Subsequently, pure state human pluripotent stem cell induction culture methods using only various chemical inhibitors in quasi-state human embryonic stem cells without gene introduction have been reported by many research groups (Chan, et al., 2013, Cell stem cell 13: 663; Gafni et al., 2013, Nature 504: 282; Ware, et al., 2014, PNAS, 111: 4484; Wang et al., 2014, Nature 516: 405; Theunissen et al., 2014, Cell Stem Cell 15: 471; Duggal et al., 2015, Stem Cells 33: 2686; Guo et al., 2016, Stem Cell Reports 6: 437; Qin et al., 2016, Cell Reports 14: 2301; Zimmerlin et al., 2016, Development 143: 4368; Guo et al., 2017, Development 144: 2748; Yang et al., 2017, Cell 169: 243). Characteristics are still debated.

Recently, there has been fierce competition to develop efficient methods for culturing human pluripotent stem cells mainly in the United States, the United Kingdom, Israel, Singapore, Japan, Germany, Australia, and Belgium, because of the many advantages of human pure pluripotent stem cells. . Human pure pluripotent stem cells can not only study embryos before implantation of humans, but can also be single-cell cultured by trypsin treatment when compared to human quasi-pluripotent stem cells, making it easy for beginners to cultivate, rapid cell growth, and high It has a differentiation ability and has the advantage of easy gene edition, so it is expected to be a new concept of pluripotent stem cell that has higher clinical value and can promote practical practical use (Collier, et al., 2018, Bioassays 40: 1700239 ). However, in the early stages of research, the characteristics of human pure pluripotent stem cells are different according to each method, so there is no standardization of the exact pure state and characteristics. Therefore, in each culture method, a pure state, a semi-state, an intermediate state, etc. are mixed, and surface markers that can be used by purely separating human pluripotent stem cells are not known (Collier, et al., 2018, Bioassays 40: 1700239). Currently, three methods are known to distinguish between pure and quasi-state human pluripotent stem cells. The method uses surface markers that are detected in the quasi-state and not in the pure-state. The first is a CD24 marker. And in the state of being used, it is used to express highly (Shakiba et al., 2015, Nature Commun. 6: 7329). The second method uses a representative quasi-state human pluripotent stem cell marker, SSEA-4, which is negative in human pure pluripotent stem cells (Pastor et al., 2016, Cell Stem Cell 18: 323). The third method is to distinguish quasi-state human pluripotent stem cells using four antibodies that recognize GPR64, CDH3, NLGN4X, and PCDH1 that do not bind to pure human pluripotent stem cells (O'Brien, et al., 2017, Stem Cells 35: 626). However, it is not easy to distinguish human pure pluripotent stem cells in the presence of various cell populations during reprogramming in the absence of a known positive surface marker for human pure pluripotent stem cells (Collier, et al., 2018, Bioassays 40: 1700239; Trusler et al., 2018, Stem Cell Research 26:36). Recently, the Peter Rugg-Gunn team at Cambridge University in England distinguished between pure and quasi-state human pluripotent stem cells using antibodies that recognize 486 cell surface markers, and is positive only for human pure pluripotent stem cells, CD7, CD75, CD77, and It was reported that the CD130 marker was found (Collier et al., 2017 Cell Stem Cell 20: 784). However, they are often negative when cultured with human pure pluripotent stem cells according to the known culture method, and in addition to the pure state, they also bind to epiblasts and extraembryonic cells in human embryos. Therefore, it is necessary to discover new surface markers specific to human pure pluripotent stem cells, because only antibodies that identify human pure pluripotent stem cells currently in use are not sufficient to analyze the characteristics of the pure state cells and separate the cells. Pure pluripotent stem cells Through the discovery of new surface markers, it can be applied to the development of a medium that induces efficient culture of pure pluripotent stem cells and the development of efficient cell therapeutics.

The object of the present invention is a monoclonal antibody N16 that recognizes the carbonic anhydrase 14 (Carbonic Anhydrase 14; CA14) protein on the surface of human pure pluripotent stem cells, which binds to human pure pluripotent stem cells and does not bind to human semi-pluripotent stem cells. -F2 is provided.

Another object of the present invention is to provide a hybridoma that produces the monoclonal antibody described above.

Another object of the present invention is to provide a human pure pluripotent stem cell assay kit comprising the monoclonal antibody described above.

Another object of the present invention is to provide a composition for purely separating human pure pluripotent stem cells containing the monoclonal antibody described above.

Another object of the present invention is to provide a method for purely separating human pure pluripotent stem cells using the monoclonal antibody described above.

Another object of the present invention is to provide a composition for removing human pure pluripotent stem cells containing the monoclonal antibody described above.

Another object of the present invention is to provide a method for removing human pure pluripotent stem cells using the monoclonal antibody described above.

1. Heavy chain complementary determine region 1 (HCDR1) comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, and heavy chain comprising HCDR3 comprising SEQ ID NO: 3; And a light chain comprising LCDR1 comprising SEQ ID NO: 4, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6, antibodies specific to naive pluripotent stem cells.

2. The antibody according to item 1, comprising a heavy chain comprising SEQ ID NO: 7 and a light chain comprising SEQ ID NO: 8.

3. The antibody according to item 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody according to item 1 or 2, wherein the antibody does not bind to a primary pluripotent stem cell.

5. The antibody according to item 1 or 2, wherein the antibody specifically binds to CA14 of human pure pluripotent stem cells.

6. The polynucleotide encoding the antibody of item 1 or 2.

7. The polynucleotide sequence according to item 6, which encodes a heavy chain complementary determine region 1 (HCDR1) comprising SEQ ID NO: 9; A polynucleotide sequence encoding HCDR2 comprising SEQ ID NO: 10; A polynucleotide sequence encoding HCDR3 comprising SEQ ID NO: 11; A polynucleotide sequence encoding LCDR1 comprising SEQ ID NO: 12; A polynucleotide sequence encoding LCDR5 comprising SEQ ID NO: 13; And a polynucleotide sequence encoding LCDR6 comprising SEQ ID NO: 14.

8. The polynucleotide according to item 6, comprising the polynucleotide sequence of SEQ ID NO: 15 encoding the heavy chain, and the polynucleotide sequence of SEQ ID NO: 16 encoding the light chain.

9. A vector comprising the polynucleotide of item 6.

10. A hybridoma producing the antibody of item 1 or 2.

11. A composition for detecting pure pluripotent stem cells, comprising the antibody of item 1 or 2.

12. The antibody of item 1 or 2; And a kit for detecting pure pluripotent stem cells.

13. A method of detecting pure pluripotent stem cells, comprising mixing the antibody of item 1 or 2 with stem cells and determining the stem cells to which the antibody is bound as pure pluripotent stem cells.

14. A method for producing an antibody that specifically binds to pure pluripotent stem cells by culturing the hybridoma of item 10.

Since the monoclonal antibody N16-F2 of the present invention specifically recognizes the cell surface protein of human pure pluripotent stem cells, a human pluripotent stem is provided by providing a tool to study the difference between early embryo development in mice and higher animals. It can be used for cell analysis and research, and can be useful for isolating or removing human pure pluripotent stem cells when developing a cell therapeutic agent, and can also provide information on the development of a serum-free medium for human embryonic stem cells.

Figure 1 is a small dome-shaped pure H9 cells (H9-2i / L / X / F /) cultured in quasi H9 (primed H9) and pure pluripotent stem cell induction medium 2i / L / X / F / P. P).

Figure 2 shows the human pluripotent stem cell markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD24, and CD90, and human pure pluripotent stem cell markers CD7, CD75, which were given through FACS analysis. CD77, and CD130 expression shown, in the converted pure pluripotent stem cells (H9-2i / L / X / F / P), all semi-pluripotent stem cell marker expression except CD90 is reduced and known as pure pluripotent stem cell marker CD7 and CD77 are pictures showing increased expression. The solid line is each monoclonal antibody, and the gray background includes only secondary antibodies.

Figure 3 is a pure pluripotent stem cell-specific gene expression pattern through quantitative polymerase chain reaction H9-2i / L / X / F / P cells, a pure pluripotent stem cell gene (Stella, Esrrb, Prdm14, Rex1) , Klf2, Klf4, Klf5 expression is significantly higher than that in semi-H9 cells, and the expression of Otx2, Sox17, and Dnmt3B, which are quasi-primitive stem genes, decreases in pure H9 cells.

Figure 4 is a monoclonal antibody of the present invention through FACS analysis N16-F2 does not bind to quasi H9, quasi-CHA-hES4, quasi-iPS-NT4-S1 cells, mouse embryonic stem cells (R1), mouse embryonic fibroblasts (MEF). In addition, in various cancer cell analysis, N16-F2 binds to human embryonic carcinoma cells NT-2, 2102Ep, NCCIT, but with 7 general cancer cells (Huh7, A375, A549, HepG2, NCI-H146, SNU387, SH-SY5Y) It shows no binding to human peripheral blood mononuclear cells (PBMC) at all. At this time, the solid line is a monoclonal antibody and the gray background is the negative control data including only the secondary antibody.

Figure 5 is a monoclonal antibody of the present invention through FACS analysis N16-F2 does not bind to quasi H9, quasi-CHA-hES4, quasi-iPS-NT4-S1 cells, and includes 2i / L / X / F / P Pure H9 cells (H9-2i / L / X / F / P, H9-2i / L / F / A, H9-LCDM) and pure CHA-hES4 (CHA-hES4-2i / L) induced by different methods / X / F / P, CHA-hES4-2i / L / F / A, CHA-hES4-LCDM). In addition, N16-F2 is another type of pluripotent stem cell induced iPS-NT4-S1, which is also a pluripotent stem cell line, and does not bind to iPS-NT4-S1, but is derived from three iPS-NT4-S1 cells (iPS -NT4-S1-2i / L / X / F / P, iPS-NT4-S1-2i / L / F / A, iPS-NT4-S1-LCDM). At this time, the solid line is a monoclonal antibody and the gray background is the negative control data including only the secondary antibody.

6 is purified by protein-G sepharose column chromatograpy in hybridoma culture medium of clone 1 (c1) and clone 2 (c2) of antibody N16-F2, and 10% SDS-PAGE The figure analyzed with HC represents the heavy chain, and LC represents the light chain.

Figure 7 after biotinylation of human embryonic carcinoma cell NT-2 cell surface (Non-F2 monoclonal antibody clones c1 and c2) and immunoprecipitated, the precipitated protein was analyzed on 10% SDS-PAGE ( Left A), again transferred to the PVDF membrane by Western blotting and analyzed by streptavidin-HRP (SA-HRP) (right B). Except for the antibody, the same experiment was performed as a negative control (No Ab), and the precipitated protein was observed around 45 kDa.

Figure 8 is a monoclonal antibody N16 of the present invention through LC-MS / MS analysis of the peptide obtained after cleaving trypsin by separating the protein immunoprecipitated from NT-2 cells with the c1 clone antibody of N16-F2 on SDS-PAGE. As a result of showing that the antigen recognized by -F2 is CA14 (Carbonic anhydrase 14), each of the peptides (red) analyzed is a result showing a match among the entire amino acid sequence of CA14.

Figure 9 is a monoclonal antibody of the present invention through LC-MS / MS analysis of the peptide obtained after cleaving trypsin by separating the protein immunoprecipitated from NT-2 cells with a c2 clone antibody of N16-F2 antibody on SDS-PAGE. As a result of repeatedly showing that the antigen recognized by N16-F2 is CA14 (Carbonic anhydrase 14), it is a result showing where each peptide (red) analyzed is matched among the entire amino acid sequence of CA14. It shows that some of the peptides analyzed were different from the c1 clone result but the same CA14.

FIG. 10 is a rabbit polyclonal anti-CA14 (GTX81537, Gene) that recognizes CA14 to confirm whether the two clones c1 and c2 of the monoclonal antibody N16-F2 of the present invention recognize CA14 protein and immunoprecipitate. Tex) This is the result confirmed by Western blot with rabbit anti-CA14 antibody after immunoprecipitation (IP) of CA14 in NT-2 cells together with the antibody. At this time, the H9 cell extract without IP was simultaneously analyzed as a positive control, and the c1 antibody of N16-F2 itself was also analyzed as a negative control. Rabbit anti-CA14 antibodies only bind to the CA14 isoform (glycosylated). The N16-F2 antibody binds better to the original form of the 45kDa than the isoform, but shows that it binds both the isotype and the 45kDa form.

11 is an expression vector for expressing CA14 protein having a myc tag in 293FT cells.

Figure 12 is a monoclonal antibody of the present invention N16-F2 to confirm the recognition of the CA14 protein by a foreign gene introduction method to prepare a vector expressing Myc-labeled CA14 and introduced into 293FT cells, CA14-myc is well (A), Myc, N16-F2 c1 and c2 antibodies, and immunoprecipitation (IP) using CA14 antibodies, and immunoprecipitated antigens were detected by rabbit anti-Myc antibodies, and c1 and c2 antibodies were Myc It shows that it recognizes CA14 like an antibody or CA14 antibody. At this time, the input was the cell extract before IP, and the N16-F2 c1 antibody and CA14 antibody were analyzed together without IP as a control.

FIG. 13 shows the heavy chain gene (HC) and light chain gene (LC) using reverse transcriptase-polymerase chain reaction after extracting total RNA from hybridoma N16-F2 two clones c1 and c2 to clone the antibody N16-F2 gene. ) Is the result of amplifying the variable region.

FIG. 14 shows the base sequence and amino acid sequence of the N16-F2 c1 and c2 antibody heavy chain gene variable regions, and is a diagram showing the complementarity determining region (CDR) binding to an antigen and residue positions of all amino acids.

FIG. 15 shows the nucleotide sequence and amino acid sequence of the variable regions of the N16-F2 c1 and c2 antibody light chain genes, and is a diagram showing the residue positions of the CDR and all amino acids that bind to the antigen.

The present invention will now be more fully described below with reference to the accompanying drawings, but only some, but not all, embodiments of the invention are illustrated. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the specific examples presented herein. The singular form used in this specification and the appended claims includes a plurality of objects unless expressly indicated otherwise.

When a monoclonal antibody against surface molecules of human pure pluripotent stem cells is prepared by directly injecting human cultured human pluripotent stem cells, which is different from the commercially available methods of producing antibodies (using peptides and recombinant proteins), It is expected to discover cell surface molecules that are specific to pluripotent stem cells and that are actually functional. The monoclonal antibody made in this way can be used to more accurately analyze the characteristics of human pure pluripotent stem cells and to purely isolate or remove human pure pluripotent stem cells in the development of cell therapeutics. And by using these antibodies to discover specific human pure pluripotent stem cell surface molecules and identifying their role in stem cell maintenance, regeneration, and differentiation ability, it can be applied to the development of new media for efficient culture of human pure pluripotent stem cells. .

Under this background, the present inventors improved and applied the bait immunization method used to excavate surface antigen CD133 of blood stem cells to prepare a group of monoclonal antibodies specific to human pure pluripotent stem cells (Choi et al., 2008, Cell and Tissue Research 333: 197). First, human quasi-pluripotent stem cells H9 (primed H9) containing 2i / L / X / F / P (MEK / GSK inhibitor, LIF, XAV939, Forskolin, Purmorphamine, KOSR, DMEM / F12 medium) (Zimmerlin et al. , 2016, Development 143: 4368), and can be cultured as a single cell by small dome-shape and trypsin treatment, and human pure pluripotent stem cell H9 (H9-2i /) that expresses highly pure pluripotent stem cell-specific genes. L / X / F / P). Quasi-H9 cells were pre-punched into the right hind paw of the mouse, and after 3 days, H9-2i / L / X / F / P cells were injected into the left hind paw, followed by a total of 7 additional immunizations at 3 day intervals, and then the ovarian lymph nodes of the left hind paw Was isolated and used to prepare monoclonal antibodies. The group of monoclonal antibodies specific to these human pure pluripotent stem cells bound to human pure pluripotent stem cells and did not bind to or weakly bound to human quasi pluripotent stem cells. We completed the present invention by confirming that these antibody populations N16-F2 antibodies recognize CA14 (Carbonic Anhydrase 14) on the surface of human pure pluripotent stem cells and show different properties from previously known antibodies.

In one embodiment, the present invention is a monoclonal antibody N16- that recognizes CA14, which is an enzyme containing zinc metal and is known as a type I membrane protein, as a new surface marker of human pure pluripotent stem cells. It's about F2.

In one specific embodiment, the monoclonal antibody N16-F2 of the present invention is a monoclonal antibody that binds to human pure pluripotent stem cells and does not bind or weakly bind to human quasi pluripotent stem cells.

In another specific embodiment, the monoclonal antibody N16-F2 of the present invention is a monoclonal antibody that does not bind to mouse embryonic stem cells and specifically recognizes human pure pluripotent stem cells.

In a preferred embodiment, the monoclonal antibody N16-F2 of the present invention is a monoclonal antibody comprising the nucleotide sequence and amino acid sequence shown in FIGS. 14 and 15.

In the present invention, the term “monoclonal antibody” refers to a protein molecule that is directed to a single antigenic site (single epitope) and specifically binds thereto. For the purposes of the present invention, the monoclonal antibody of the present invention is a protein molecule that specifically recognizes the cell surface molecule of human pure pluripotent stem cells because it specifically binds to the cell surface molecule of human pure pluripotent stem cells.

Since the main regions of antibodies that recognize antigen-specific epitopes and form antigen-antibody complexes are variable regions of the heavy and light chains, particularly complementarity determining regions (CDRs), these inventions contribute to the formation of the complex. The variable region of the monoclonal antibody of the present invention, in particular its chimeric antibody comprising a CDR, humanized antibody and the like are included in the scope of the present invention. In addition, the present invention includes functional fragments of antibody molecules as well as complete forms with 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 means a fragment having at least an antigen-binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.

The present inventor used an improved bait immunization method to produce monoclonal antibodies specific to human pure pluripotent stem cells (Choi et al., 2008, Cell and Tissue Research 333: 197). Specifically, after confirming the expression of flat colonies and undifferentiated markers, which are typical cell patterns of quasi H9 cells (FIGS. 1 and 2A), these quasi H9 cells are first injected into the right side of the mouse's hind paw as a bait immunogen, and 2i / L / X / F Antibody against surface antigens commonly present in both cells by injecting dome-shaped human pure pluripotent stem cells H9-2i / L / X / F / P cells (Figs. 1 and 2B) induced by / P medium to the left. They were collected and removed from the right hind paw lymph node, and lymphocytes of the left lymph node containing only antibodies specific to human pure pluripotent stem cells were isolated to prepare a monoclonal antibody group, and among them, the antigen was analyzed using N16-F2 antibody. .

Specifically, semi-H9 cells were cultured in a medium containing 2i / L / X / F / P and 5% oxygen concentration to confirm the formation of dome-shaped colonies within 3-5 days, and among the characteristics of human pure pluripotent stem cells After confirming that it is possible to culture into single cells at intervals of 3 to 4 days by one trypsin treatment, a quantitatively specific gene (STELLA, ESRRB, PRDM14, REX1) was used using a quantitative analysis polymerase chain reaction (qRT-PCR). KLF2 / 4/5) was confirmed to be highly expressed in cells cultured in 2i / L / X / F / P medium (FIG. 3). The prepared bait immunogen (semi-H9 cells) and the real immunogen (H9-2i / L / X / F / P cells) were used to prepare a monoclonal antibody group that specifically binds to human pure pluripotent stem cells.

Subsequently, the cultured quasi-H9 cells and H9-2i / L / X / F / P cells were detached with collagenase IV, and then immunized through the soles of the hind legs of the mouse, and the left ovarian lymph node. The monoclonal antibody group, which is a specific embodiment of the present invention, was separated from hybridomas prepared by fusion of lymphocytes and fusion with FO myeloma cells to isolate lymphocytes, of which 85 antibodies were H9-2i / L / X / It was confirmed to bind to the F / P cell surface. Again, it was confirmed that 14 of them also bind to CHA-hES4-2i / L / X / F / P cells, a pure pluripotent stem cell type derived from another human quasi pluripotent stem cell CHA-hES4 cell. It was also confirmed that it did not bind to cells (R1), mouse embryonic fibroblasts (MEF), and the like. Among these antibodies, the N16-F2 antibody is a monoclonal antibody with IgG1 and kappa chains, binds to human pure pluripotent stem cells and human embryonic carcinoma cells, and human semi-pluripotent stem cells, mouse embryonic stem cells, mouse embryonic fibroblasts, 7 species It was confirmed that it does not bind to various cancer cells, and human peripheral blood monocytes (PBMC) (Fig. 4, 5). Specifically, in order to further verify whether the antibody N16-F2 shows specific binding to various human pure pluripotent stem cells, a recent method 2i / L / F / A of the method of converting human pluripotent stem cell H9 cells to pure pluripotent stem cells Medium (MEK / GSK inhibitor, LIF, Forskolin, Ascorbic acid, KOSR, KO-DMEM) (Duggal et al., 2015, Stem Cells 33: 2686) B, or LCDM medium (LIF, GSK inhibitor, Dimethundene maleate, Minocycline hydrochloride , IWR-1-endo, N2 / B27, KOSR, DMEM / F12) (Yang et al., 2017, Cell 169: 243) -derived analysis for binding to other human pure pluripotent stem cells Again confirmed (FIG. 5), another human quasi pluripotent stem cell CHA-hES4 cells were converted to pure pluripotent stem cell in three different ways (CHA-hES4-2i / L / X / F / P, CHA -hES4-2i / L / F / A, CHA-hES4-LCDM) was also confirmed (Fig. 5). In addition, in other forms of human pluripotent stem cells, iPS-NT4-S1, induced pluripotent stem cell states, various pure pluripotent stem cell states (iPS-NT4-S1-2i / L / X / F / P) do not bind. , iPS-NT4-S1-2i / L / F / A, iPS-NT4-S1-LCDM) is a highly practical antibody that recognizes the binding of N16-F2 as a universal marker of human pure pluripotent stem cells. It was confirmed (Fig. 5).

Therefore, in order to verify reproducibility, N16-F2 antibodies c1 and c2 were purified from a hybridoma culture medium producing N16-F2 antibody using two clones c1 and c2 of the N16-F2 hybridomas (FIG. 6), respectively. Both antibodies were used in subsequent experiments simultaneously. Using these antibodies, N16-F2 c1 and c2 clone antibodies were confirmed to immunoprecipitate about 45 kDa cell surface protein (Fig. 7). The protein molecular weight of human pure pluripotent stem cells recognized by the monoclonal antibody of the present invention was measured using 10% SDS-PAGE, and there may be some errors within a certain range depending on the measurement conditions of the molecular weight. Therefore, the use of the term “about” in avoiding the molecular weight of a protein cannot be avoided, and may generally have a range of ± 2 kDa, preferably ± 1 kDa.

The proteins recognized by c1 and c2 of the monoclonal antibody N16-F2 were immunoprecipitated, separated from SDS-PAGE, and then cut with trypsin to confirm that the protein was CA14 by LC-MS / MS analysis (Fig. 8, Fig. 9). ). Rabbit polyclonal CA14 antibody after immunoprecipitation using N16-F2 c1 and c2 clones and commercially available rabbit polyclonal CA14 antibody to verify that the antigen recognized by the c16 and c2 antibodies of N16-F2 is CA14 After Western blotting, it was confirmed that the antigen recognizing N16-F2 was CA14 (FIG. 10), and the vector expressing the CA14 gene linked with Myc tag (FIG. 11) was transfected with 293FT cells, and myc tagged It was confirmed that CA14 was expressed at the expected position (FIG. 12A), and immunoprecipitation using rabbit Myc, N16-F2-c1, N16-F2-c2, and rabbit CA14 antibodies was confirmed by Western blotting (FIG. 12B). ). Accordingly, the genes of the N16-F2 clones c1 and c2 antibodies were cloned by the polymerase chain reaction method (FIG. 13), and the respective heavy chain and light chain nucleotide sequences and amino acid sequences were identified to obtain CA14 having the same heavy chain light chain amino acid sequence. It was confirmed that the recognition is a new mouse monoclonal antibody (Fig. 14, 15).

CA14 recognized by N16-F2, a monoclonal antibody of the present invention, is known as an enzyme (zinc metalloenzyme) containing zinc metal that catalyzes the reversible hydration of carbon dioxide, and is a membrane protein present in the cell surface membrane. (Mboge et al., 2018 Metabolites 8:19; Singh, S et al., 2018 Molecules 23: 1045). Carbonic anhydrase plays an important role in ion transport, acid-base balance, gas exchange, photosynthesis, and CO ₂ fixation. 15 isoforms are known (Supuran CT 2016, Biochem. J. 473: 2023; Mboge et al., 2018 Metabolites 8:19).

Eight of them (CA 1, 2, 3, 4, 8, 10, 11, 13) are present in the cytoplasm, 2 (CA 5A, 5B) are mitochondria, and 1 (CA 6) is secreted out of the cell, 4 Dogs (CA 4 (GPI binding), 9, 12, 14) are bound to the membrane. Among membrane-bound CAs, CA9 and CA12 have been shown to play an important role in cancer development, cancer cell signaling, cancer progression and metastasis, and acidification, and many chemical inhibitors or antibodies against them have been developed (Supuran CT 2017, Metabolites 7:48; Singh, S et al., 2018 Molecules 23: 1045). CA14 of the membrane-bound CA has one transmembrane region, and was found in mice in 1999 (Mori, Ogawa et al. 1999, JBC 274: 15701), but soon human CA14 was also identified (Fujikawa-Adachi, Nishimori et al. 1999, Genomics 61: 74), not much known compared to other membrane binding CAs.

CA14 is expressed in the membranes and axons of neurons present in the brains of humans and mice (Seppo et al., 2001, PNAS 98: 1918), and is also expressed in human pluripotent stem cells. However, nothing is known about its function (Sperger, et al., 2003 PNAS 100: 13350). According to a recently reported study, EGR1, EOMES, EWS-ERG, FOXA2, GATA1 / 2, MITF, NANOG, SOX2, TRIM28, etc. are representative transcription factors that bind to the promoter of the human CA14 gene (Rouillard AD et al. ., Database (oxford), 2016, 2016: baw100). NANOG, SOX2, and TRIM28 are representative transcription factors that regulate the starch and self-renew ability of human pure and semi-pluripotent stem cells, and the remaining transcription factors are starch differentiation potentials such as NANOG, SOX2, and OCT4 during embryogenesis. It is known as a differentiation factor that is directly regulated by a regulator. Therefore, CA14 expression in human pure pluripotent stem cells is thought to be closely regulated by starch potential transcription factors such as NANOG, SOX2, and TRIM28. The expression of CA14 on the surface of human pure pluripotent stem cells can serve as an important cell surface marker that defines human pure pluripotent stem cells by first revealing with the use of the specific monoclonal antibody N16-F2 of the present invention. It can be used to isolate and analyze pure human pluripotent stem cells. In addition, through the antibody sequencing of the monoclonal antibody N16-F2 of the present invention, it was proved that it is a new antibody with a different antibody sequence from the antibody against CA14 developed before the present invention.

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 present invention provides a hybridoma that produces monoclonal antibody N16-F2.

In a specific embodiment, the hybridomas of the present invention are injected with H9-2i / L / X / F / P cells and semi-H9 cells into the left and right soles of the mice, respectively, and then separate the lymphocytes from the left sediment of the mice and myeloma. It was prepared by fusion with cancer cells.

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

The monoclonal antibody produced by the above hybridoma may be used without purification, but in order to obtain the best results, it is purified and used with high purity (for example, 95% or more) according to a method well known in the art. It is desirable to do. Such a purification technique may be separated from the culture medium or ascites fluid using, for example, purification methods such as gel electrophoresis, dialysis, salt precipitation, and chromatography.

In a specific embodiment of the present invention, for mass production of the monoclonal antibody of the present invention, the culture medium of hybridomas was purified using protein G-sepharose column chromatography.

In another aspect, the present invention relates to a composition for isolating human pure pluripotent stem cells comprising the monoclonal antibody N16-F2 described above.

In another aspect, the present invention relates to a method for isolating human pure pluripotent stem cells using the monoclonal antibody N16-F2 described above.

In another aspect, the present invention relates to a composition for removing human pure pluripotent stem cells comprising the monoclonal antibody N16-F2 described above.

In another aspect, the present invention relates to a method for removing human pure pluripotent stem cells using the monoclonal antibody N16-F2 described above.

The monoclonal antibody group of the present invention can be used to isolate high-efficiency human pure pluripotent stem cells present in a mixed cell population for the development of an efficient cell therapy agent.

The monoclonal antibody group of the present invention can be used to remove human pure pluripotent stem cells present in cells to be transplanted for cell therapy.

To selectively remove human pure pluripotent stem cells, the monoclonal antibody of the present invention can be coupled (eg, covalently) with a known therapeutic agent either directly or indirectly through a linker or the like. Therapeutic agents that can be combined with the antibody include, but are not limited to, radionuclides, drugs, lymphokines, toxins, and release-type antibodies.

To remove human pure pluripotent stem cells present in the transplanted cells, the antibody can be administered by itself or in a composition comprising the antibody. In the case of a composition comprising an antibody, it is prepared in an appropriate formulation, including an acceptable carrier depending on the mode of administration. Formulations suitable for the mode of administration are known in the art. These formulations are administered by suitable methods including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal and intra-lesion administration where necessary for local immunosuppressive therapy. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferred modes of administration and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. The composition comprising the antibody of the present invention can be administered in a pharmaceutically effective amount to remove human pure pluripotent stem cells. Typical dosage levels can be optimized using standard clinical techniques.

In another aspect, the present invention relates to an assay kit for human pure pluripotent stem cells comprising the monoclonal antibody described above.

The monoclonal antibody of the present invention can be used not only to remove human pure pluripotent stem cells in transplanted or transplanted cells through an antigen-antibody complex reaction, but also to specifically detect human pure pluripotent stem cells.

Such assay kits include monoclonal antibodies of the invention, as well as tools, reagents, and the like commonly used in the art for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling agents capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, and the like.

When the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction stopper may be included. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, Polymers such as polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.

Formation of antigen-antibody complexes includes tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), western blotting, immunoprecipitation assay, immunodiffusion assay, and complement fixation Analysis methods (Complement Fixation Assay), FACS, protein chips, and the like, but are not limited thereto.

Labels that enable qualitative or quantitative measurement 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 that can be used as detection labels include β-glucuronidase, β-glucosidase, β-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and GDPase, These include RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphenolpyruvate decarboxylase, and β-lactamase. It is not limited. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoerytherin, phycocyanin, allopicocyanine, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. The emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. The microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, 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.

The term "vector" in the present invention includes both viral carriers as well as non-viral carriers for introducing nucleic acids into cells in vitro, ex vivo or in vivo. The vector may be a replicon with another DNA fragment attached to amplify the attached fragment.

A “replicon” is any genetic element (eg, plasmid, phage, cosmid, chromosome, that can act as an autonomous unit of DNA replication in vivo, ie capable of replication under its own control), Virus). A large number of vectors known in the art can be used to manipulate nucleic acids, incorporate response elements and promoters into genes, and the like. Preferred vectors include, for example, plasmids or modified viruses comprising, for example, adenovirus, retrovirus, adeno-associated virus, herpes virus, or plasmids such as pBR322 or pUC plasmid derivatives or Bluescript vectors. It includes. For example, insertion of a DNA fragment corresponding to a reaction element and a promoter into an appropriate vector can be accomplished by combining the appropriate DNA fragment with a selected vector having a complementary cohesive termini. Otherwise, the ends of the DNA molecule can be enzymatically modified, or any site can be created by binding the nucleotide sequence (linker) to the DNA ends. Such vectors can be engineered to contain a selection marker gene for selecting cells that incorporate markers into the cell genome. Such markers enable identification and / or selection of host cells expressing by incorporating the protein encoded by the marker.

Vectors provide essential regulatory sequences (eg, transcriptional and translational elements) to regulate the expression of fusion proteins in appropriate host cells. Regulatory sequences include promoter region, enhancer region, transcription termination site, ribosome binding site, initiation codon, splice signal, intron, polyadenylation signal, Shine / Dalgarno translation sequence and Kozak consensus sequence. sequence). The regulatory sequence is selected taking into account the host cell in which the fusion protein will be produced. Suitable bacterial promoters include, but are not limited to, bacteriophage λpL or pR, T6, T7, T7 / lacO, lac, recA, gal, trp, ara, hut and trp-lac. Suitable eukaryotic promoters include PRBI, GAPDH, metallothionein, thymidine kinase, viral LTR, cytomegalovirus, SV40, or tissue-specific or tumor-specific promoters such as α-fetoprotein, amylase, cathepsin E, M1 muscarinic receptor or γglutamyl transferase.

Additional vectors include lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes) and protein-DNA complexes. In addition to nucleic acids, vectors can also include one or more regulatory regions, and / or selectable markers useful for selecting, measuring and monitoring nucleic acid delivery results (delivery to any tissue, duration of expression, etc.).

The term "selection marker" in the present invention is an identification factor that can be selected based on the effect of the marker gene, that is, antibiotic resistance, herbicide resistance, colorimetric marker, enzyme, fluorescent marker, etc., generally an antibiotic or chemical resistance gene, Here, the effect is used to track the inheritance of the desired nucleic acid and / or to identify cells or organisms that have inherited the desired nucleic acid. Examples of selectable marker genes known and used in the art include ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, and the like; And phenotypic markers, that is, anthocyanin regulatory genes, isopentanyl transferase genes, and the like.

In one embodiment of the invention, the vectors introduced into the cell further include a "selection marker gene" indicating that the vector was introduced into the host cell when expressed. In this way, the selector gene can be a positive marker for the presence of a vector. Although not critical to the method of the present invention, the presence of a selection marker gene allows the experimenter to select a living cell population into which the vector construct has been introduced into the cell. Thus, certain embodiments of the invention include selecting cells for which the vector has been successfully introduced. As used herein, the term “screening” or variations thereof is intended to mean a well-known standard method of selecting cells with a particular genetic matrix or phenotype when used with cells. Typical methods include, but are not limited to, culturing cells in the presence of antibiotics such as G418, puromycin and ampicillin. Other examples of selectable marker genes include, but are not limited to, genes that confer resistance to methotrexate, hygromycin, or mycophenolic acid. Cells containing an antibiotic resistant gene or a vector construct comprising genes will be able to withstand the antibiotic in culture. Likewise, cells that do not contain an antibiotic resistant gene or vector construct containing genes will not be able to withstand the antibiotic in culture.

The antibody of the present invention can detect pure pluripotent stem cells by combining with an imaging marker. Administration and identification of antibody-imaging marker conjugates and methods of linking antibodies to imaging markers (Goldenberg et al., 1978, New England J. Med. 298: 1384; Goldenberg et al., 1983) , J. Amer.Med.Assoc. 280: 630; Goldengerg et al., 1983, Gastroenterol. 84: 524; Siccardi et al., 1986, Cancer Res. 46: 4817; Epenetos et al., 1985, Cancer 55: 984; Philben et al., 1986, Cancer 57: 571; Chiou et al., 1986, Cancer Inst. 76: 849; Colcher et al., 1983, Cancer Res., 43: 736; Colcher, E. et al. , Laboratory Research Methods in Biology and Medicine Immunodiagnostics.New York, Alan R. Liss.pp.215-258 (1983); Keenan, AM et al., 1984, J. Nucl.Med. 25: 1197; Colcher D. et al., 1987, Cancer Res. 47: 1185; Estaban, JM et al., 1987, Intl. J. Cancer 39:50; Martin, DT, et al., 1984, Curr. Surg. 41: 193; Martin, EW Jr. et al., 1986, Hybridoma 5: S97; Martin, DT et al., 1985, Am. J. Surg. 150: 672; Meares et al., Anal. Biochem. 1984, 142: 68; and Krejcarek et al. , 1977, Biochem. And Biophys. Res. Comm. 77: 581). The dosage may vary depending on the amount of pluripotent stem cells. The antibody-imaging marker conjugate dosage should be an effective amount to visualize or confirm pure pluripotent stem cells from semi-pluripotent stem cells.

Examples of imaging markers capable of binding to antibodies are well known to those skilled in the art and use a gamma scanner or hand held gamma probe or a proton emission tomograpy. Therefore, it includes a material that can be identified by diagnostic imaging and a material that can be identified by nuclear magnetic resonance imaging using a nuclear magnetic resonance spectrometer or the like. Suitable examples of materials that can be identified by gamma scanners, etc. are ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹¹¹ In, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, and ^99m Radioactive isotopes such as Tc. ¹²⁵ I, ¹²³ I, ¹⁵³ Sm and ^{99 m} Tc are preferred because of their low energy and extensive identification. Gadolinium (Gd) is an example of a material that can be identified using a nuclear magnetic resonance spectroscopy.

In the present invention, the terms "nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are used interchangeably, and ribonucleosides in single-stranded form or double-stranded helix (adenosine, guanosine) , Uridine or cytidine; "RNA molecule") or polymer form of phosphate esters of deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymine or deoxycytidine; "DNA molecule") or Refers to any phosphoric acid ester analogue thereof, such as phosphorothioate and thioester. Double helix DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Thus, the term includes, among others, linear or circular DNA molecules (eg, restriction enzyme fragments), plasmids, supercoiled DNA and double-stranded DNA found as chromosomes. When discussing the structure of a specific double-stranded DNA molecule, sequences are described herein according to the general convention of presenting the sequence only in the 5 'to 3' direction along the untranscribed DNA strand (i.e., a strand having a sequence matching the mRNA). This can be described. A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA and semisynthetic DNA.

In the present invention, the term “instruction” means verbal instructions (eg, verbal instructions from a doctor, veterinarian, health professional, professional seller or sales organization) and / or radio or television media (ie advertising), or written instructions (eg For example, written instructions from a doctor, veterinarian, or other health professional (e.g., a prescription), written instructions from a professional seller or sales organization (e.g., a marketing booklet, brochure or other instructional facility), written media (e.g. For example, it may include instructions that may be on the Internet, electronic mail, or other computer-related media. The manual may be in various forms, including, for example, paper, computers, personal digital assistants (PDAs), telephones (including mobile phones and other communication devices) or other devices used to communicate voice or text, the Internet, etc. And / or may be included and / or stored and / or delivered.

<Example 1> Culture of human pluripotent stem cells and establishment of human pure pluripotent stem cells

<1-1> Culture of human quasi-pluripotent stem cells and mouse embryonic stem cells

Human quasi pluripotent stem cell H9 was cultured according to the protocol provided by the Wicell Research Institute, and CHA-hES4 and human induced pluripotent stem cell line iPS-NT4-S1 were cultured by pre-sale at Cha Hospital. In embryonic CF1 mice, embryonic fibroblasts (MEFs) were extracted and cultured and used as feeder cells after γ irradiation. 20% (v / v) KOSR (knockout serum replacement), 1% (v / v) NEAA, 0.1 mM β-mercaptoethanol, 100 U / mL penicillin-G, 100 μg / mL streptomycin, Cultured in DMEM / F12 (Invitrogen) medium with 8 ng / ml bFGF added, and passaged every 5-6 days with collagenase IV (1 mg / ml) for 5 minutes, followed by an appropriate size with a yellow tip Cut into and incubated on fresh feeder cells. Mouse embryonic stem cell R1 is 15% (v / v) FBS, 0.1 mM β-mercaptoethanol, 1 mM glutamine, 0.1 mM NEAA, 100 U / mL penicillin-G, 100 μg / mL streptomycin, 500 units / mL on supporting cells The cells were cultured using DMEM medium (Invitrogen) composed of LIF (leukemia inhibitory factor, PeproTech). Human peripheral blood mononuclear cells (PBMC) were isolated using the method suggested by the Ficoll-Paque Plus method (GE Healthcare, Seoul, Korea).

<1-2> Establishment of human pure pluripotent stem cells

Zune H9, Zune CHA-hES4, Zune iPS-NT4-S1 cells were treated with collagenase IV (1 mg / ml) for 5 minutes, cut to appropriate size using a yellow tip and transferred onto pre-laid MEFs. Next day 2i / L / X / F / P medium (1 μM PD0325901, 3 μM CHIR99021, hLIF (20 ng / ml), 4 μM XAV939, 10 μM Forskolin, 2 μM Purmorphamine, 20% (v / v) KOSR, DMEM / F12, 1% ( v / v) L-glutamine, 1% (v / v) NEAA, 0.1 mM β-mercaptoethanol, 1X penicillin / streptomycin) (Zimmerlin et al., 2016, Development 143: 4368), 2i / L / F / A medium (1 μM PD0325901, 3 μM CHIR99021, hLIF (1000 U / ml), 10 μM Forskolin, ascorbic acid (50 ng / ml), bFGF (16 ng / ml), 20% (v / v) KOSR, KO-DMEM, 1% (v / v) L-glutamine, 1% (v / v) NEAA, 0.1 mM β-mercaptoethanol, 1X penicillin / streptomycin) (Duggal et al., 2015, Stem Cells 33: 2686), or LCDM media (10 ng / ml hLIF, 1 μM CHIR99021, 2 μM (S)-(+)-Dimethundene Maleate, 2 μM Minocycline hydrochloride, 0.5-1 μM IWR-1-endo, 2 μM Y-27632, N2 / B27, 1% (v / v) GlutaMAX, 1% (v / v) NEAA, 0.1 mM β-mercaptoethanol, 5% (v / v) KOSR, DMEM / F12) (Yang et al., 2017, Cell 169: 243). Human pure pluripotent stem cells cultured in a 5% (v / v) oxygen incubator in 2i / L / X / F / P medium turn into Dome-shaped cells after 3-5 days, and established pure pluripotent stem cells H9 cells The cells were passaged using 0.05% (v / v) trypsin-EDTA at 3-4 day intervals (FIG. 1).

<1-3> Cell surface antigen staining

In order to compare human quasi-pure and pure pluripotent stem cells, the expression of SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24, and CD90, known as human quasi-potential stem cell markers, was first analyzed by FACS. For FACS analysis, cells separated with collagenase IV were separated into single cells using TrypLE (Invitrogen), and then passed through a 40 μm strainer, and about 2 to 3 X 10 ⁵ cells per sample were used. After reacting SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24-PE, and CD90-PE antibodies at 4 ° C for 30 minutes, PBA (1% (w / v) bovine serum albumin, 0.02% (w / v) after washing twice with NaN ₃ in PBS), the primary antibody and the corresponding anti-rat IgM-FITC, anti-mouse IgM-FITC or anti-mouse IgG-FITC (BD Biosciences) at 30 ° C. It reacted for a minute more. After washing twice with PBA, FACSCalibur and Cell Quest software (BD sciences) were used to analyze antibody binding to PI (propidium iodide) -negative cells. In the same way, CD7, CD75, CD77, and CD130 expressions known as pure pluripotent stem cell markers were also observed. As a result, it was observed that the expressions of SSEA3, SSEA4, TRA-1-60, TRA-1-81, and CD24, excluding CD90, among human quasi pluripotent stem cell markers were significantly reduced in human pure pluripotent stem cell markers, and were pure pluripotent stem cell markers. CD7 and CD77 expressions known as H9-2i / L / X / F / P increased, confirming that human pure pluripotent stem cells were well induced (FIGS. 2A and 2B).

<1-4> Pure pluripotent stem cell specific gene expression confirmation

Human quasi and pure pluripotent stem cells were separated by collagenase IV treatment, reacted for 30 minutes in a 0.1% (w / v) gelatin-coated plate to remove support cells, and then total RNA was analyzed using RNAiso Plus (TaKaRa). Was extracted. After synthesizing cDNA using PrimeScript RT Master Mix (TaKaRa), the expression of pure and semi-specific genes was confirmed through qRT-PCR using Power SYBR Green PCR Master Mix (Applied Biosystem). At this time, primer sequences for amplifying the used gene are listed in Table 1.

As a result, in pure H9 cells derived from 2i / L / X / F / P medium, the expression of Stella, Esrrb, Prdm14, Rex1, Klf2, Klf4, and Klf5, which are pure pluripotent stem cell genes, was significantly more pronounced than in the H9 cells. Increasing and expressing the pluripotent stem cell genes (semigenes) Otx2, Sox17 and Dnmt3B was significantly reduced in pure H9 cells, confirming that human pure pluripotent stem cells were well induced in 2i / L / X / F / P medium. (Fig. 3).

<Example 2> Preparation of hybridoma

<2-1> Decoy immunization for the production of human pure pluripotent stem cell specific antibody

To create a monoclonal antibody that specifically binds only to the cell surface and internal molecules of pure H9 cells, 2X10 ⁶ quasi H9 cells -3, 0, 3, 6 were added to the right plantar of 11 female Balb / c mice, 6 weeks old. , 13, 17, 20, 21 days, and the same number of pure H9-2i / L / X / F / P cells were injected into the left foot sole on 0, 3, 6, 13, 17, 20, 21 days, respectively. On the 22nd day, the immunized mice were dislocated from the cervical spine, and then the popliteal lymph nodes were removed from the left hind paw to remove fat and muscle tissue. The surface of the surface was frosted slide glass. It was prepared by grinding single lymph nodes using a single cell. Left lymph node cells and FO myeloma cells are mixed at a ratio of 1: 5, washed twice with DMEM (Invitrogen) without serum, and then 1 ml 50% (w / v) PEG1500 (polyethylene glycol, BMS, seoul) , Korea) to mix 25 drops per minute. After 1 minute, 3 ml of DMEM medium was continuously added for 1 minute, 17 ml for 1 minute, and 20 ml for 1 minute. After standing for 5 minutes, the cells were collected by centrifugation. 2X10 ⁵ cells / well in a 96-well plate was cultured for about 2 weeks in DMEM medium containing 20% (v / v) FBS, HAT, and HCF (hybridoma cloning factor) for about 2 weeks, and a hybrid that produced antibodies using a sandwich ELISA method Only chopping boards were selected.

<2-2> Cloning of hybridomas

Sandwich Enzyme Linked Immunosorbent Assay (ELISA) method was used to select clones expressing the antibody. 100 μl of hybridoma culture was added to a plate coated with 2 μg / ml of an anti-mouse IgG or IgM antibody, which is a capture antibody, and reacted at 37 ° C. for 1 hour, and again anti-mouse IgG-HRP or anti -Reacted for 1 hour with 1 / 5,000 dilution of mouse IgM-HRP (horseradish peroxidase, Sigma). After washing three times with PBST (0.05% (v / v) Tween-20 in PBS), a substrate solution containing OPD (o-phenylenediamine, Sigma) and H ₂ O ₂ was added, and absorbance at 492 nm was measured. Thus, clones producing antibodies were first selected. Next, a hybridoma that binds H9-2i / L / X / F / P cells to the culture medium of each clone through FACS analysis described in Example <1-3> is selected and subcloned, A group of 33 hybridomas secreting a group of monoclonal antibodies that maintained binding to H9-2i / L / X / F / P cells was selected. Twelve of these antibodies were further selected by binding to CHA-hES4-2i / L / X / F / P cells, and double antibodies N16-F2 were antibodies with IgG1 and κ chains, and the recognition antigens and properties were analyzed in this patent. .

<Example 3> Analysis of binding specificity of monoclonal antibody N16-F2

<3-1> Binding specificity analysis for human semi- and pure pluripotent stem cells, mouse embryonic stem cells, various cancer cells, and differentiated normal cells

FACS analysis was performed to observe the degree of binding of the monoclonal antibody N16-F2 to various cells. Specifically, quasi H9, quasi CHA-hES4, quasi iPS-NT4-S1, mouse embryonic stem cells (R1), mouse embryonic fibroblasts (MEF), various cancer cell lines, human peripheral blood mononuclear cells (PBMC) and three methods The human induced pluripotent stem cells prepared with are prepared, and the pluripotent stem cells are first treated with collagenase Ⅳ㎍ / ml) for 5 minutes to detach the cells, washed twice with PBS (pH 7.4), and then TrypLE The solution (Invtrogen) was added and reacted at room temperature for 3 minutes, and mouse embryonic stem cells R1 and various cancer cells were removed by treatment with 0.05% (w / v) trypsin-EDTA. To remove all the separated cells, filter them using a 40 μm strainer (BD Biosciences, Seoul, Korea) to separate them into single cells, mix each single cell with approximately 2X10 ⁵ cells per ml in PBA, and then Example <1. The monoclonal antibody N16-F2 was reacted at 4 ° C for 30 minutes in the same manner as -3>. After washing twice with PBA, the primary antibody and the corresponding anti-mouse IgG-FITC (BD Biosciences) were further reacted at 4 ° C. for 30 minutes. After washing twice with PBA, antibody binding capacity was analyzed for propidium iodide (PI) -negative cells using FACSCalibur and Cell Quest software (BD sciences). In Figure 4, the solid line indicates the binding force of N16-F2, and the gray background includes only the secondary antibody. N16-F2 did not bind to quasi-H9, quasi-CHA-hES4, quasi-induced pluripotent stem cells iPS-NT4-S1 cells, mouse embryonic stem cells (mESC R1), mouse embryonic fibroblasts (MEF), and 7 different types It also did not bind to cancer cells, but did not bind to human embryonic carcinoma cells NT-2, 2102EP, NCCIT, and human peripheral blood mononuclear cells (PBMC), which are differentiated human normal cells (Fig. 4).

On the other hand, N16-F2 specifically binds to three types of H9 pure pluripotent stem cells (H9-2i / L / X / F / P, H9-2i / L / F / A, H9-LCDM), and CHA-hES4 It showed specific binding ability to pure pluripotent stem cells (CHA-hESC4-2i / L / X / F / P, CHA-hESC4-2i / L / F / A, CHA-hES4-LCDM), and pure induced pluripotent stem cells The binding force to iPS-NT4-S1 (iPS-NT4-S1-2i / L / X / F / P, iPS-NT4-S1-2i / L / F / A, iPS-NT4-S1-LCDM) induced by Showed. Therefore, it was confirmed that N16-F2 is a highly practical antibody that recognizes surface antigens that are universally expressed in various types of human pure pluripotent stem cells (FIG. 5).

<3-2> Purification of monoclonal antibody N16-F2

To characterize the monoclonal antibody N16-F2, the antibody was purified in N16-F2 hybridoma. For reproducibility and accuracy of characterization, hybridoma cell cultures of two subclones, clone c1 and clone c2 cells derived from N16-F2 hybridoma, were independently collected and purified and tested for the same two antibodies respectively. And observed whether the same experimental result can be obtained. First, the cell culture solution was recovered and centrifuged at 3,000 rpm for 5 minutes to use only the supernatant. Protein-G Sepharose was added to the chromatography column, the column was connected to a peristatic pump, washed sufficiently with PBS (pH8), and slowly passed through the antibody supernatant to pass c1 and c2 antibodies to Protein-G Sepha It was bound to the Rose column. After sufficiently washing with PBS (pH8), the antibody was eluted with 0.2M glycine (pH 2.7), and 1 M Tris-HCl (pH 9.0) was added to neutralize the eluate. This antibody was used after dialysis in PBS. The purified antibody amount was measured using a BCAM protein assay kit (BCATM Protein Assay Kit, Pierse), and the purification pattern was confirmed using SDS-PAGE to confirm that the N16-F2 antibodies were purified from c1 and c2 clones, respectively. (Fig. 6).

<Example 4> Identification of antigens recognized by monoclonal antibody N16-F2

<4-1> Identification of cell surface protein molecules recognized by c1 and c2 of monoclonal antibody N16-F2

A method of producing an antibody that specifically binds to pure pluripotent stem cells by culturing the hybridoma of claim 10 to which the N16-F2 antibody binds.

Biotinylation of the cell surface was performed by slightly modifying the protocol presented by EZ-Link Sulfo-NHS-LC-Biotin (Pierce). NT-2 cells cultured in a 100 mm cell culture plate were washed twice with PBS (pH 7.4) preheated at 37 ° C., and then cold PBS (pH 8.0) in which Biotin (0.5 mg / ml) was dissolved was added, and 4 It was reacted at ℃ for 30 minutes. After washing 3 times with cold PBS (pH 8.0), Biotin labeled cells were lysed buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% (v / v) Nonidet P-40, 2 μg / ml After reacting for 30 minutes at 4 ° C with aprotinin, 100µg / ml PMSF, and 5µg / ml leupeptin, 1mM NaF, 1mM Na ₃ VO ₄ ), centrifuged at 12,000rpm for 40 minutes to remove the nucleus. Stored at -70 ° C until below.

In order to remove the protein non-specifically binding to protein-G agarose (Santa Cruz Biotechnology, Santa Cruz, CA), 20 μl protein-G agarose was added to the cell lysate of approximately 1 × 10 ⁷ cells and 2 at 4 ° C. After reacting for a period of time, the protein-G agarose was recovered through centrifugation, and a sample washed 4-5 times using lysis buffer was used as a negative control. In order to immunoprecipitate antigens recognized by c1 and c2 of N16-F2, 2 μg of each antibody was added to the cell lysate from which protein non-specifically binding to protein-G agarose was removed, reacted at 4 ° C. for 12 hours, and then 20 After adding µL protein-G agarose, the mixture was further reacted at 4 ° C for 2 hours. The immunoprecipitated immune mixture was washed 4-5 times using a lysis buffer, and 5X sample buffer was added to elute the antigen bound to each antibody, and heated at 100 ° C for 5 minutes. The eluted protein was separated using 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and stained with Instant Blue (Expedeon) to detect that c1 and c2 specific proteins (N16-F2 antigen) were detected at about 45 kDa. It was confirmed (Fig. 7A).

The negative control protein and the eluted protein were separated using 10% SDS-PAGE, followed by western blotting with nitrocellulose membrane. The membrane was blocked for 1 hour at room temperature using 5% (w / v) skim milk powder. After washing three times with 0.1% (v / v) PBST, streptavidin-HRP (1: 7,000; GE healthcare) was further reacted at room temperature for 1 hour. The cells were washed three times with 0.1% (v / v) PBST, and the biotin-labeled cell surface protein was confirmed by an ECL detection kit (GE healthcare). As a result, c1 and c2 of the monoclonal antibody N16-F2 were confirmed to immunoprecipitate about 45 kDa cell surface protein, respectively (Fig. 7B).

<4-2> Identification of antigens immunoprecipitated by c1 and c2 of monoclonal antibody N16-F2

SDS gels containing proteins immunoprecipitated by c1 and c2 of the monoclonal antibody N16-F2 were stained with Instant Blue staining solution (Expedeon) according to the supplier's protocol (Fig. 7A). The protein was enzymatically digested into small fragments using modified porcine trypsin modified according to the method of Shevchenko (Shevchenko, et al., Anal. Chem. 68: 850-858, 1996). It was washed with 50% (v / v) acetonitrile to remove impurities such as SDS, organic solvent, and dye reagent from the gel pieces. Then, it was reacted with trypsin (8-10 ng / µl) at 37 ° C. for 8-10 hours. Proteolysis was terminated by addition of 5 μl 0.5% trifluoroacetic acid. The protein fragments cut by trypsin were recovered in an aqueous solution, and desalted and concentrated to a volume of 1-5 µl using C18ZipTips (Millipore). This concentrate was mixed with α-cyano-4-hydroxycinnamic acid saturated in 50% aqueous acetonitrile in the same amount and dropped onto the target plate for mass spectrometry. The mass spectrometer was Ettan MALDI-TOF (Amersham Biosciences). The protein fragments loaded on the target plate were vaporized by 337nm N2 laser irradiation, and then accelerated by a 20Kv injection pulse. The mass spectrum for each protein spot was determined by the cumulative peak of 300 laser shots. For the analysis of the mass spectrum, the ion peak m / z (842.510, 2211.1046) of the peptide produced by autolysis of trypsin was used as a standard peak. For the identification of proteins from the mass spectrum of which analysis was completed, a ProFound search engine (http://129.85.19.192/profound_bin/ WebProFound.exe) developed by Rockefeller University was used. As a result, it was confirmed that c1 and c2 recognize the same CA14 (Carbonic Anhydrase 14) (FIGS. 8 and 9).

<Example 5> monoclonal antibody N16-F2 antigen verification

<5-1> Verification of CA14 antigen immunoprecipitated by monoclonal antibody N16-F2

To confirm whether the CA14 protein immunoprecipitated by the monoclonal antibody N16-F2 is recognized by a commercially available CA14 antibody, human embryonic carcinoma NT-2 cell extract was added to two N16-F2 clone antibodies c1. After immunoprecipitation with c2 and rabbit polyclonal CA14 (GTX81537, Gene Tex) antibodies, the negative control protein and the eluted protein were separated using 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked for 1 hour at room temperature using 5% (w / v) skim milk powder. After washing three times with 0.1% (v / v) PBST, rabbit polyclonal CA14 (N-term) antibody was reacted at 4 ° C. for 12 hours. After washing three times with 0.1% (v / v) PBST, anti-rabbit IgG-HRP (1: 10,000) was further reacted at room temperature for 1 hour. After washing three times with 0.1% (v / v) PBST, it was confirmed with an ECL detection kit (GE healthcare). As a result, it was observed that the two clones c1 and c2 of the monoclonal antibody N16-F2 of the present invention recognize the CA14 protein like the rabbit polyclonal anti-CA14 antibody that recognizes CA14 and cause immunoprecipitation ( Fig. 10). At this time, it was observed that the rabbit anti-CA14 antibody binds only to the CA14 isoform (glycosylated), and the N16-F2 antibodies, c1 and c2, are better at the original form of CA14 at 45 kDa than the glycosylated isoform. It has been shown to bind, but can bind to both isoforms of CA14 (Figure 10).

<5-2> Verification of N16-F2 antigen by myc-tagged CA14 gene expression

In order to confirm that the antigen of the monoclonal antibody N16-F2 is CA14, the pCMV-CA14 (hMU004339, KHGB) vector was purchased and EcoRI (F); After PCR using 5'-CCGAATTCATGTTGTTCTCCGCCCTCCT-3 'and CA14-XbaⅠ5'-GGTCTAGATGCCTCAGTCGTGGCTT-3' primers, PCR products and pcDNA3.1 (+)-Myc / His vector (Invitrogen) were used as EcoRⅠ and XbaⅠ enzymes. After treatment and ligation, a pcDNA3.1 (+)-CA14-Myc / His vector was prepared (FIG. 11). This vector expressing CA14 was transfected with 293FT cells using a PEI solution, and then normal expression of Myc-tagged CA14 was confirmed with an anti-Myc tag (Abcam) antibody (FIG. 12A). Once again, the pcDNA3.1 (+)-CA14-Myc / His vector was transfected into 293FT cells, reacted for 30 minutes at 4 ° C using the lysis buffer specified in Example <4-1>, and then at 40 at 12,000 rpm. Immunoprecipitation (IP) of Myc-tagged CA14 with anti-Myc tag, N16-F2 c1 and c2 clone antibody, rabbit anti-CA14 antibody using supernatant (1 mg) from which the nucleus was removed by centrifugation for 1 minute As a result of confirming that the precipitated antigen is detected by the anti-Myc antibody, the antigen immunoprecipitated by the anti-Myc, N16-F2-c1, N16-F2-c2, and anti-CA14 antibodies is detected by the anti-Myc antibody. Was confirmed. (Figure 12B). These results demonstrate that the N16-F2 antibody recognizes Myc-tagged CA14, thereby repeatedly proving that the antigen recognized by the N16-F2 antibody is CA14.

<Example 6> Monoclonal antibody N16-F2 antibody gene and amino acid analysis

<6-1> Gene cloning of N16-F2 c1 and c2 clone antibodies

After harvesting 5 x 10 ⁶ centrifugal crops of each hybridoma N16-F2 cell c1 and c2 clones, the total RNA was extracted with RNA iso plus reagent (TaKaRa, Otsu, Japan). . The obtained total RNA was quantified by measuring A260. Total RNA was added to Prime Script RT Master Mix (TaKaRa) to prepare a reverse transcriptase chain reaction mixture to synthesize cDNA. To clone the antibody gene, a known polymerase chain reaction primer was modified and used (Wang, et al 2000, J. Immunol. Methods 233: 167). For heavy chain cloning with the synthesized cDNA, the polymerase chain reaction primer corresponding to the IgG1 constant region, the base sequence 5'-GGA GTC GAC ATA GAC AGA TGG GGG TGT CGT TTT GGC-3 ', an oligonucleotide 25 pmole and a heavy chain antibody variable region Primer corresponding to N-terminal sequence 5'MH1 5'-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TC-3 'and 5'MH2- 5'-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TCW GG- 3 'phosphorus oligonucleotide was added to prepare a chain polymerization reaction mixture. For light chain cloning, 5'- ggt gtc gac GGA TAC AGT TGG TGC AGC ATC-3 'oligonucleotide which is a primer corresponding to the kappa chain constant region and 5'MK 5'-cgg which is the primer corresponding to the N terminus of the kappa chain variable region aag ctt GAY ATT GTG MTS ACM CAR WCT MCA-3 'and Primer 6 5'- GAC ATT GTG CTG ACC CAA TCT CCA GCT TCT-3' and Primer 7 5'-GAC ATT CAG CTG ACC CAG TCT CCA-3 'respectively Was used. For efficient cloning of the polymerase chain reaction product, SalI restriction site was assigned to the 3'-primer end of the light chain, and HindIII restriction site was assigned to the 5'-primer. In the heavy chain, EcoRI was assigned to the 5'-primer and SalI restriction enzyme was assigned to the 3'-primer. After mixing the heavy chain and light chain reaction solutions, the reaction was performed 30 times at 95 ° C for 1 minute, 58 ° C for 45 seconds, and 72 ° C for 1 minute. As a result, amplified DNA was obtained at a position corresponding to a length of about 400 bp, which is estimated to be a DNA fragment corresponding to the heavy chain constant region, and about 390 bp, a length estimated to be a DNA fragment corresponding to the light chain constant region (FIG. 13).

<6-2> Cloning and sequencing of genes of N16-F2 c1 and c2 clone antibodies

In order to clone the c1 and c2 antibody genes of N16-F2 amplified in the above example, the polymerase chain reaction product was first treated with EcoRI and SalI for the heavy chain, and 1.0% (w / v) after treatment with HindIII and SalI for the light chain. The DNA corresponding to about 400 bp and 390 bp was isolated using a FavorPrep GEL ™ PCR Purification Kit (Favorgen, Taiwan) by deploying it on an agarose gel. PBluescript KS + to be used as a vector to clone the heavy chain gene was treated with EcoRI and SalI, and pBluescript KS + was treated with HindIII and SalI as a light chain gene cloning vector, and then separated with a FavorPrep GEL ™ PCR Purification Kit (Favorgen, Taiwan). These two DNAs were linked with T4 DNA ligase (New England Biolab, USA), transformed into E. coli DH5α by CaCl ₂ method, and cloned with a DNA insert of about 400 bp in size for heavy chains and about 390 bp in size for light chains E. coli clones were selected.

For DNA sequencing of antibody genes, the above several clones were cultured overnight in 3 ml of LB medium containing 100 µg / ml of ampicillin, and then plasmid DNA was isolated using a DNA miniprep kit (Intron, Korea), respectively. The nucleotide sequence of the DNA insert was determined. After changing the base sequence of heavy and light chain cDNA to amino acids, each amino acid sequence was analyzed using the Kabat database (Johnson G. and Wu, T.T. 2001, Nucleic Acids Res. 29: 205). The gene sequences obtained from the c1 and c2 clones were identical to each other, confirming that they were identical antibodies, and the numbers above the base sequences of the heavy chain sequences (FIG. 14) and light chain sequences (FIG. 15) were determined according to Kabat numbering. As a result of analyzing each amino acid sequence, these immunogenes have residues and arrangements characteristic for the antibody structure (Figs. 14 and 15). Specifically, among several groups of immunoglobulins, the heavy chain belongs to the subgroup IIA, and the light chain As a CDR residue that recognizes the antigen, CDR1 is 23-35, CDR2 is 50-66, CDR3 is 99-104, and CDR1 is 24 ~ 39, CDR2 is 55 ~ for light chain. 62, CDR3 corresponded to 94-102. The structurally essential disulfide bonds involved cysteines 22 and 96 for the heavy chain and cysteines 23 and 93 for the light chain. From the analysis results of this gene, it was confirmed that the heavy and light chain genes are functional.

While the invention has been presented and described above with reference to some embodiments, those skilled in the art will readily appreciate that the specific experiments described above are merely illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. For example, in the above assays, examples have been described according to the activity of an antibody, but it will be understood that one or more variants, e.g., an antibody having the same CDR sequence and at least 85% sequence identity, have the described activity. . Other variants may be similar, have less activity, have no activity, or may have some antagonistic activity when assayed for function.

For example, for purposes of claim construction, the claims set forth below should not be interpreted in any way narrower than their literal language, and therefore, exemplary implementations from the specification should not be read as claims. Accordingly, it should be understood that the invention has been described by way of illustration and not limitation on the scope of the claims. Accordingly, the invention is limited only by the following claims. All publications, patents, patent applications, books, and journal articles cited in this application are hereby incorporated by reference in their entirety.

<110> Sejong University Industry-Academy Cooperation Group

<120> Monoclonal antibody N16-F2 specific to human naive pluripotent

         stem cells

<130> 18OP10022PCT

<160> 16

<170> KoPatentIn 3.0

<210> 1

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 1

Lys Ala Ser Gly Tyr Thr Leu Thr Asp Tyr Ser Met His

  1 5 10

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 2

Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys

  1 5 10 15

Gly

<210> 3

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 3

Gly Arg Glu Arg Gly Phe

  1 5

<210> 4

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 4

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr

  1 5 10 15

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 5

Gln Met Ser Asn Leu Ala Ser

  1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 6

Ala Gln Asn Leu Glu Leu Pro Tyr Thr

  1 5

<210> 7

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> HC full sequence

<400> 7

Gln Val Lys Leu Glu Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

  1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Asp Tyr

             20 25 30

Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

         35 40 45

Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

     50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Ile Ala Tyr

 65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

                 85 90 95

Thr Arg Gly Arg Glu Arg Gly Phe Trp Gly Gln Gly Thr Thr Leu Thr

            100 105 110

Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

        115 120

<210> 8

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> LC full sequence

<400> 8

Asp Ile Val Met Thr Gln Ser Thr Phe Ser Asn Pro Val Thr Leu Gly

  1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

             20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

         35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

     50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

                 85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

            100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser

        115 120

<210> 9

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> HCDR1

<400> 9

aaggcttctg gttataccct cacagactat tcaatgcac 39

<210> 10

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> HCDR2

<400> 10

tggataaaca ctgagactgg tgagccaaca tatgcagatg acttcaaggg a 51

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> HCDR3

<400> 11

ggacgcgaaa ggggattc 18

<210> 12

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> LCDR1

<400> 12

aggtctagta agagtctcct acatagtaat ggcatcactt atttgtat 48

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> LCDR2

<400> 13

cagatgtcca accttgcctc a 21

<210> 14

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> LCDR3

<400> 14

gctcaaaatc tagaacttcc gtacacg 27

<210> 15

<211> 372

<212> DNA

<213> Artificial Sequence

<220>

<223> HC full sequence

<400> 15

caagttaagc tggagcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cttctggtta taccctcaca gactattcaa tgcactgggt gaagcaggct 120

ccaggaaagg gtttaaagtg gatgggctgg ataaacactg agactggtga gccaacatat 180

gcagatgact tcaagggacg gtttgccttt tctttggaaa cctctgccag cattgcctat 240

ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtac tagaggacgc 300

gaaaggggat tctggggcca aggcaccact ctcacggtct cctcagccaa aacgacaccc 360

ccatctgtct at 372

<210> 16

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> LC full sequence

<400> 16

gatattgtga tgacacaatc tacattctcc aatccagtca ctcttggaac atcagcttcc 60

atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120

tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180

tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240

agcagagtgg aggctgagga tgtgggtgtt tattactgtg ctcaaaatct agaacttccg 300

tacacgttcg gaggggggac caagctggaa ataaaacggg ctgatgctgc accaactgta 360

tcc 363

Claims (14)

Heavy chain complementary determine region 1 (HCDR1) comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, and heavy chain comprising HCDR3 comprising SEQ ID NO: 3; And a light chain complementary determine region 1 (LCD1) comprising SEQ ID NO: 4, an LCDR2 comprising SEQ ID NO: 5, and a light chain comprising LCDR3 comprising SEQ ID NO: 6. Antibodies specific to naive pluripotent stem cells. The antibody of claim 1, comprising a heavy chain comprising SEQ ID NO: 7 and a light chain comprising SEQ ID NO: 8. The antibody according to claim 1 or 2, wherein the antibody is a monoclonal antibody. The antibody according to claim 1 or 2, wherein the antibody does not bind to a primary pluripotent stem cell. The antibody according to claim 1 or 2, wherein the antibody specifically binds to carbonic anhydrase 14 (CA14) of pure pluripotent stem cells. A polynucleotide encoding the antibody of claim 1 or 2. The method according to claim 6, Heavy chain complementary determining region 1 (SEQ ID NO: 9) comprising a polynucleotide sequence encoding a complementary determine region 1 (HCDR1); A polynucleotide sequence encoding HCDR2 comprising SEQ ID NO: 10; A polynucleotide sequence encoding HCDR3 comprising SEQ ID NO: 11; A polynucleotide sequence encoding LCDR1 comprising SEQ ID NO: 12; A polynucleotide sequence encoding LCDR5 comprising SEQ ID NO: 13; And a polynucleotide sequence encoding LCDR6 comprising SEQ ID NO: 14. The polynucleotide according to claim 6 or 7, comprising the polynucleotide sequence of SEQ ID NO: 15 encoding a heavy chain, and the polynucleotide sequence of SEQ ID NO: 16 encoding a light chain. A vector comprising the polynucleotide of claim 6 or 7. A hybridoma producing the antibody of claim 1 or 2. A composition for detecting pure pluripotent stem cells, comprising the antibody of claim 1 or 2. A kit for detecting pure pluripotent stem cells, comprising the antibody of claim 1 or 2. A method of detecting pure pluripotent stem cells, comprising mixing the antibody of claim 1 or 2 with stem cells and determining the stem cells to which the antibody is bound as pure pluripotent stem cells. A method for producing an antibody that specifically binds to pure pluripotent stem cells by culturing the hybridoma of claim 10.

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