ANTIBODY FOR PURE ISOLATION OF VASCULAR ENDOTHELIAL CELLS AND PREPARATION METHOD THEREOF

Abstract

The present specification provides an antibody for pure isolation of vascular endothelial cells and a preparation method thereof, the antibody comprising: a heavy chain variable domain and a light chain variable domain which specifically bind to an extracellular matrix domain of PECAM1 having an amino acid sequence of SEQ ID NO: 1; or a heavy chain variable domain and a light chain variable domain which specifically bind to an extracellular matrix domain of CDH5 having an amino acid sequence of SEQ ID NO: 2.

Claims

1. An antibody for purifying a vascular endothelial cell, the antibody containing a heavy-chain variable domain and a light-chain variable domain specifically binding to an extracellular matrix domain of PECAM1 having an amino acid sequence represented by SEQ ID NO: 1 or CDH5 having an amino acid sequence represented by SEQ ID NO: 2.

2. The antibody of claim 1, wherein the antibody includes a monoclonal antibody specifically binding to the extracellular matrix domain of PECAM1 or the extracellular matrix domain of CDH5.

3. The antibody of claim 1, wherein the antibody further includes a heavy-chain invariable domain and a magnetic particle, wherein the magnetic particle is attached to the heavy-chain invariable domain of the antibody.

4. A method for producing an antibody for purifying a vascular endothelial cell, the method comprising: injecting a protein containing an extracellular matrix domain of PECAM1 having an amino acid sequence represented by SEQ ID NO: 1 into an antibody producing subject so as to produce an antibody against the PECAM1; determining a positive clone reacting with an antigen of the PECAM1 from the antibody producing subject; and separating the antibody against the PECAM1 from the positive clone.

5. The method of claim 4, wherein the method further includes, prior to the injection of the protein into the antibody producing subject: preparing a recombinant plasmid vector containing a base sequence expressing the extracellular matrix domain of the PECAM1; transfecting the recombinant plasmid vector into a host cell; and obtaining the protein containing the extracellular matrix domain of the PECAM1.

6. The method of claim 4, wherein the determining of a positive clone includes: separating a plurality of B lymphocytes from the antibody producing subject; fusing the plurality of B lymphocytes with myeloma cells respectively to produce fused cells; culturing the plurality of the fused cells respectively to form a clone; and determining the positive clone to react with the antigen of the PECAM1 from the formed clone, wherein separating the antibody includes purifying a monoclonal antibody against the PECAM1 from the positive clone.

7. The method of claim 4, wherein a concentration of the separated antibodies against the PECAM1 is at least 3.5 mg/ml.

8. The method of claim 4, wherein the separated antibodies against the PECAM1 maintain a specificity and a binding affinity to the extracellular matrix domain of the PECAM1 at a concentration of 0.004 mg/ml or lower of the antibodies.

9. A method for producing an antibody for purifying a vascular endothelial cell, the method comprising: injecting a protein containing an extracellular matrix domain of CDH5 having an amino acid sequence represented by SEQ ID NO: 2 into an antibody producing subject so as to produce an antibody against the CDH5; determining a positive clone reacting with an antigen of the CDH5 from the antibody producing subject; and separating the antibody against the CDH5 from the positive clone.

10. The method of claim 9, wherein the method further includes, prior to the injection of the protein into the antibody producing subject: preparing a recombinant plasmid vector containing a base sequence expressing the extracellular matrix domain of CDH5; transfecting the recombinant plasmid vector to a host cell; and obtaining the protein containing the extracellular matrix domain of CDH5.

11. The method of claim 9, wherein the determining of the positive clone includes: separating a plurality of B lymphocytes from the antibody producing subject; fusing the plurality of B lymphocytes with myeloma cells respectively to produce fused cells; culturing the plurality of the fused cells respectively to form a clone; and determining the positive clone to react with the antigen of CDH5 from the formed clone, wherein separating the antibody includes purifying a monoclonal antibody against the CDH5 from the positive clone.

12. The method of claim 9, wherein a concentration of the separated antibodies against the CDH5 is at least 3.5 mg/ml.

13. The method of claim 9, wherein the separated antibodies against the CDH5 maintain a specificity and a binding affinity to the extracellular matrix domain of the CDH5 at a concentration of 0.004 mg/ml or lower of the antibodies.

14. A method for purifying a vascular endothelial cell, the method comprising: differentiating an endothelial cell from a stem cell to obtain a cell cluster containing a vascular endothelial cell and an endothelial cell different from the vascular endothelial cell; applying the antibody of one of claims 1 to 3 to the cell cluster, such that a protein of PECMA1 or a protein of CDH5 present on a surface of the vascular endothelial cell immuno-responses to the antibody for purifying the vascular endothelial cell; and sorting a cell exhibiting fluorescence in an immunofluorescence staining process due to the immuno-response as a vascular endothelial cell.

15. The method of claim 14, wherein the stem cell includes a human pluripotent stem cell or a human induced pluripotent stem cell.

16. The method of claim 14, wherein the differentiating includes culturing the stem cell in a DLL4-treated medium to differentiate the endothelial cell.

17. The method of claim 14, wherein the antibody for purifying the vascular endothelial cell further contains a magnetic particle, wherein the sorting includes: permeating the antibody-treated cell cluster into a column, wherein the column receives a metallic particle therein, and a magnetic force is externally applied to the column; and sorting a cell bound to the metallic particle located inside the column as the vascular endothelial cell.

18. The method of claim 17, wherein the sorting includes sorting the vascular endothelial cell by blocking the magnetic force externally applied to the column.

19. A kit for purifying a vascular endothelial cell, the kit comprising the antibody of one of claims 1 to 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1A illustrates a procedure of the method of preparation of an antibody against PECAM1 for purifying vascular endothelial cells according to an embodiment of the present disclosure.

[0065] FIG. 1B shows the extracellular matrix domains of PECAM1 used as antigens in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0066] FIG. 1C shows the expression levels for proteins that include extracellular matrix domains of PECAM1 used as antigens in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0067] FIG. 1D shows the level of binding affinity with antigens as measured for each of the isolated multiple of clones in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0068] FIG. 1E shows the results of immunofluorescence staining analysis for each of the isolated multiple of clones in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0069] FIG. 1F illustrates a procedure of the method of preparation of an antibody against CDH5 for purifying vascular endothelial cells according to an embodiment of the present disclosure.

[0070] FIG. 1G shows the extracellular matrix domains of CDH5 used as antigens in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0071] FIG. 1H shows the expression levels for proteins that include extracellular matrix domains of CDH5 used as antigens in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0072] FIG. 1I shows the level of binding affinity with antigens as measured for each of the isolated multiple of clones in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0073] FIG. 1J shows the results of immunofluorescence staining analysis for each of the isolated multiple of clones in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0074] FIG. 2A illustrates a procedure of the method of purifying a vascular endothelial cell using an antibody against PECAM1 for purifying the vascular endothelial cell according to another embodiment of the present disclosure.

[0075] FIG. 2B illustrates a procedure of the method of purifying a vascular endothelial cell using an antibody against CDH5 for purifying the vascular endothelial cell according to another embodiment of the present disclosure.

[0076] FIG. 3A shows the results of immunofluorescence staining analysis for each of the clones determined as multiple of positive clones reacting with PECAM1 in the preparation method of an antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0077] FIG. 3B shows the results of immunofluorescence staining for antibodies obtained by the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0078] FIG. 4A and FIG. 4B show the results of evaluation of cell specificity using immunofluorescence staining method for antibodies obtained by the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0079] FIG. 5A shows the results of immunofluorescence staining analysis for each of the clones determined as multiple of positive clones reacting with CDH5 in the preparation method of an antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0080] FIG. 5B shows the results of evaluation using immunofluorescence staining for antibodies obtained by the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0081] FIG. 6A and FIG. 6B show the results of evaluation of cell specificity using immunofluorescence staining process for antibodies obtained by the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

BEST MODES FOR CARRYING OUT THE INVENTION

[0082] Advantages and features of the present disclosure and methods of achieving them will be apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present disclosure is not limited to the embodiments disclosed below, but may be implemented in various forms. The present embodiments merely allow the disclosure of the present disclosure to be complete and are provided to completely inform the scope of the invention to those of ordinary skill in the technical field to which the present disclosure belongs. The present disclosure is only defined by the scope of the claims.

[0083] Hereinafter, referring to FIG. 1A to FIG. 1J, a method for preparing an antibodies against PECAM1 and/or CDH5 for purifying vascular endothelial cells will be described in detail. FIG. 1A illustrates a procedure of the method of preparation of an antibody against PECAM1 for purifying vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1B shows the extracellular matrix domains of PECAM1 used as antigens in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1C shows the expression levels for proteins that include extracellular matrix domains of PECAM1 used as antigens in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1D shows the level of binding affinity with antigens as measured for each of the isolated multiple of clones in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1E shows the results of immunofluorescence staining analysis for each of the isolated multiple of clones in the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1F illustrates a procedure of the method of preparation of an antibody against CDH5 for purifying vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1G shows the extracellular matrix domains of CDH5 used as antigens in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1H shows the expression levels for proteins that include extracellular matrix domains of CDH5 used as antigens in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1I shows the level of binding affinity with antigens as measured for each of the isolated multiple of clones in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 1J shows the results of immunofluorescence staining analysis for each of the isolated multiple of clones in the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0084] Referring to FIG. 1A, a preparation method of the antibody for purifying vascular endothelial cells according to one embodiment of the present disclosure may include injecting a protein containing an extracellular matrix domain of PECAM1 into the antibody producing subject to produce an antibody against PECAM1 (S110), determining a positive clone to react with the PECAM1 antigen from the antibody producing subject (S120), and separating the antibody against PECAM1 from the positive clone (S130).

[0085] More specifically, referring to FIG. 1B, in step S110 of injecting a protein containing an extracellular matrix domain of PECAM1 into an antibody producing subject, a protein having 28th to 681th extracellular matrix domains among the whole protein domain of PECAM1 may be used as an antigen and be injected into an antibody producing subject, for example, a mouse.

[0086] In this connection, the protein used as the antigen may be obtained by preparing a recombinant plasmid vector containing a base sequence expressing the extracellular matrix domain of PECAM1, transfecting the recombinant plasmid vector into the host cell, and purifying the protein including the extracellular matrix domain of PECAM1 expressed from the host cell. For example, referring to FIG. 1C, the expression level for the obtained protein of the extracellular matrix domain (ECD) of PECAM1 (CD31) as stained by coomassie blue staining is shown. The purified protein of the extracellular matrix domain of PECAM1 may be used as an antigen injected into the antibody producing subject.

[0087] Next, in step S120 of determining a positive clone, it may be obtained a positive clone in which an antibody is formed in an antibody producing subject according to an immune response. For example, referring to FIG. 1D, 51 clones determined as positive clones are shown. The 51 clones are determined as positive clones in serum isolated from mice by ELISA assay. The 51 clones have specificity to the antigen of PECAM1 among the antigens of human PECAM1 (hCD31-Fc) and the human immunoglobulin G antigen (hIgG-Fc), and react with the antigen of the PECAM1 at a high binding affinity. In this connection, the 51 positive clones exhibited high specificity and reactivity with the antigen of human PECAM1. Thus, an antibody against the antigen as the protein of the extracellular matrix domain of PECAM1 may be formed in each of the 51 positive clones.

[0088] In one example, the numerically high levels of binding affinity of the antigen-antibody may not mean that the binding affinity with the extracellular matrix domain of PECAM1 on the surface of vascular endothelial cells is substantially high at the cellular level. Therefore, according to another embodiment of the present disclosure, in the step of determining a positive clone (S120), the immunofluorescence staining method may further determine the positive clone including the antibody actually having high binding affinity with the extracellular matrix domain of PECAM1 in the cell. For example, referring to FIG. 1E, the results of performing the immunofluorescence staining method on human umbilical vein endothelial cells (HUVEC) as a human vascular endothelial cell in order to select a clone that can actually accurately detect the position of PECAM1 expression in vascular endothelial cells among the 51 clones as determined as positive clones via ELISA are shown. In this connection, among the 51 positive clones, 3F10, 4F1, 4H2, 6A3, 6A5, 6B2, 6C6, 8E5, 8E9 and 8H10 appear to fluoresce more strongly than other clones. In other words, clones of 3F10, 4F1, 4H2, 6A3, 6A5, 6B2, 6C6, 8E5, 8E9 and 8H10 were found to actually have high binding affinity with the extracellular matrix domain of PECAM1 in the human vascular endothelial cell and thus, finally, may be determined as a highly reactive positive clone.

[0089] According to another embodiment of the present disclosure, the step of determining a positive clone (S120) may include isolating a plurality of B lymphocytes from the antibody producing subject, fusing the plurality of B lymphocytes with myeloma cells respectively to produce fused cells, culturing each of the plurality of fused cells to form a clone, and determining the positive clone that reacts with the antigen of PECAM1 from the formed clone.

[0090] Finally, separating the antibody against PECAM1 (S130) may include culturing positive clones in large quantities, purifying antibodies against PECAM1, more specifically humanized PECAM1 monoclonal antibodies from the clones, thereby to obtain antibodies for purifying vascular endothelial cells.

[0091] According to another embodiment of the present disclosure, the step of isolating the antibody against PECAM1 (S130) may include passing the positive clone obtained in the step of determining the positive clone (S120) through an equilibrated column to remove the secondary proteins, and passing the protein-removed positive clone through the PECAM1 antigen-coated column to purify PECAM1 antigen-specific antibodies that may be used as antibodies for purifying the vascular endothelial cells in accordance with the present disclosure.

[0092] The PECAM1 antigen-specific antibodies as obtained by the above method may be directly applied to, in particular, ischemic tissues. Alternatively, the PECAM1 antigen-specific antibodies may be used for purifying vascular endothelial cells used in the cell therapeutic agent that may be used for the treatment of such diseases.

[0093] According to another embodiment of the present disclosure, the antibody against CDH5 for purifying vascular endothelial cells can be produced by flowing steps.

[0094] Referring to FIG. 1F, a preparation method of the antibody for purifying vascular endothelial cells according to one embodiment of the present disclosure may include injecting a protein containing an extracellular matrix domain of CDH5 into the antibody producing subject to produce an antibody against CDH5 (S110), determining a positive clone to react with the CDH5 antigen from the antibody producing subject (S120), and separating the antibody against CDH5 from the positive clone (S130).

[0095] More specifically, referring to FIG. 1G, in step S110 of injecting a protein containing an extracellular matrix domain of CDH5 into an antibody producing subject, a protein having 48th to 599th extracellular matrix domains among the whole protein domain of CDH5 may be used as an antigen and be injected into an antibody producing subject, for example, a mouse.

[0096] In this connection, the protein used as the antigen may be obtained by preparing a recombinant plasmid vector containing a base sequence expressing the extracellular matrix domain of CDH5, transfecting the recombinant plasmid vector into the host cell, and purifying the protein including the extracellular matrix domain of CDH5 expressed from the host cell. For example, referring to FIG. 1H, the expression level for the obtained protein of the extracellular matrix domain (ECD) of CDH5 as stained by a coomassie blue staining is shown. The purified protein of the extracellular matrix domain of CDH5 may be used as an antigen injected into the antibody producing subject.

[0097] Next, in step S120 of determining a positive clone, it may be obtained a positive clone in which an antibody is formed in an antibody producing subject according to an immune response. For example, referring to FIG. 1I, 43 clones determined as positive clones are shown. The 43 clones are determined as positive clones in serum isolated from mice by ELISA assay. The 43 clones have specificity to the antigen of CDH5 among the antigens of human CDH5 (hCDH5-Fc) and the human immunoglobulin G antigen (hIgG-Fc), and react with the antigen of the CDH5 at a high binding affinity. In this connection, antigen of human immunoglobulin G was used as a control antigen. The 43 positive clones exhibited high specificity and reactivity with the antigen of human CDH5 in contrast to the immunoglobulin G antigen of the control group. Thus, an antibody against the antigen as the protein of the extracellular matrix domain of CDH5 may be formed in each of the 43 positive clones. More specifically, the greater the difference between the OD value measured for the antigen of immunoglobulin G and the OD value measured for the antigen of human CDH5, the higher the binding affinity of the antigen-antibody.

[0098] In one example, the numerically high levels of binding affinity of the antigen-antibody may not mean that the binding affinity with the extracellular matrix domain of CDH5 present on the surface of vascular endothelial cells is substantially high at the cellular level. Therefore, according to another embodiment of the present disclosure, in the step of determining a positive clone (S120), the immunofluorescence staining method may further determine the positive clone including the antibody actually having high binding affinity with the extracellular matrix domain of CDH5 in the cell. For example, referring to FIG. 1J, the results of performing the immunofluorescence staining method on human umbilical vein endothelial cells (HUVEC) as a human vascular endothelial cell in order to select a clone that can actually accurately detect the position of CDH5 expression in vascular endothelial cells among the 43 clones as determined as positive clones via ELISA are shown. In this connection, among the 43 positive clones, 1A5, 3G9, 6A11, 6C9, 8D2, 9G1, 9G4, 9H10, 10D6 and 10E11 appear to fluoresce more strongly than other clones. In other words, clones of 1A5, 3G9, 6A11, 6C9, 8D2, 9G1, 9G4, 9H10, 10D6 and 10E11 were found to actually have high binding affinity with the extracellular matrix domain of CDH5 in the human vascular endothelial cell and thus, finally, may be determined as a highly relative positive clone.

[0099] According to another embodiment of the present disclosure, the step of determining a positive clone (S120) may include isolating a plurality of B lymphocytes from the antibody producing subject, fusing a plurality of B lymphocytes with myeloma cells respectively to produce fused cells, culturing each of the plurality of fused cells to form a clone, and determining the positive clone that reacts with the antigen of CDH5 from the formed clone.

[0100] Finally, separating the antibody against CDH5 (S130) may culturing positive clones in large quantities, purifying antibodies against CDH5, more specifically humanized CDH5 monoclonal antibodies from the clones, thereby to obtain antibodies for purifying vascular endothelial cells.

[0101] According to another embodiment of the present disclosure, the step of isolating the antibody to CDH5 (S130) may include passing the positive clone obtained in the step of determining the positive clone (S120) through an equilibrated column to remove the secondary proteins, and passing the protein-removed positive clone through the CDH5 antigen-coated column to purify CDH5 antigen specific antibodies that may be used as antibodies for purifying the vascular endothelial cells in accordance with the present disclosure.

[0102] The CDH5 antigen-specific antibodies as obtained by the above method may be directly applied to, in particular, ischemic tissues. Alternatively, the CDH5 antigen-specific antibodies may be used for purifying vascular endothelial cells used in the cell therapeutic agent that may be used for the treatment of such diseases.

[0103] Hereinafter, referring to FIG. 2A and FIG. 2B, a method for purifying a vascular endothelial cell using an antibody against PECAM1 or against CDH5 for purifying a vascular endothelial cell according to an embodiment of the present disclosure will be described in detail. FIG. 2A illustrates a procedure of the method of purifying a vascular endothelial cell using an antibody against PECAM1 for purifying the vascular endothelial cell according to another embodiment of the present disclosure. FIG. 2B illustrates a procedure of the method of purifying a vascular endothelial cell using an antibody against CDH5 for purifying the vascular endothelial cell according to another embodiment of the present disclosure.

[0104] Referring to FIG. 2A, the purifying method of the vascular endothelial cell according to another embodiment of the present disclosure may include differentiating the endothelial cells from the stem cells to obtain a cell cluster including the vascular endothelial cells (S210), applying the antibody for purifying the vascular endothelial cells to the cell cluster (S220), and sorting the vascular endothelial cells based on the level of fluorescence according to the immunofluorescence staining method (S230).

[0105] According to another embodiment of the present disclosure, in the step of differentiating the endothelial cell (S210), a human pluripotent stem cell or a human induced pluripotent stem cell may be used as the stem cell. Furthermore, differentiation of endothelial cells can be initiated by culturing the stem cells in a medium treated with DLL4 (notch signaling ligand).

[0106] According to another embodiment of the present disclosure, the cell cluster obtained in the step of differentiating the endothelial cells (S210) may include the inner cell mass of blastocysts, early stage embryos, cord cells, cord blood, human induced pluripotent stem cells, bone marrow, undifferentiated stem cells, stem cells of mesoderm lineage, together with endothelial cells differentiated from stem cells. However, the present is not limited thereto.

[0107] According to another embodiment of the present disclosure, the antibody used for purifying a vascular endothelial cell may further include magnetic particles. Accordingly, the step of sorting (S230) may include permeating a cell cluster to which an antibody for purifying vascular endothelial cells is applied into a column receiving metallic particles therein, in which magnetic force is externally applied to the column, and may include sorting cells bound to the metallic particles located inside the column as the vascular endothelial cells.

[0108] According to another feature of the present disclosure, the step of sorting (S230) may include sorting the vascular endothelial cells by blocking the magnetic force applied from an outside of the column.

[0109] The vascular endothelial cells isolated, at the high purity, in this way are directly used for treatment of ischemic tissues, or may be used as a cell therapeutic agent for treatment of secondary induced diseases such as cardiovascular vessel diseases such as ischemic heart disease, arteriosclerosis, myocardial infarction and angina pectoris.

[0110] Referring to FIG. 2B more, the purifying method of the vascular endothelial cell according to another embodiment of the present disclosure may include differentiating the endothelial cells from the stem cells to obtain a cell cluster including the vascular endothelial cells (S210), applying the antibody for purifying the vascular endothelial cells to the cell cluster (S220), and sorting the vascular endothelial cells based on the level of fluorescence according to the immunofluorescence staining method (S230).

[0111] According to another embodiment of the present disclosure, in the step of differentiating the endothelial cell (S210), a human pluripotent stem cell or a human induced pluripotent stem cell may be used as the stem cell. Furthermore, differentiation of endothelial cells can be initiated by culturing the stem cells in a medium treated with DLL4 (notch signaling ligand).

[0112] According to another embodiment of the present disclosure, the cell cluster obtained in the step of differentiating the endothelial cells (S210) may include the inner cell mass of blastocysts, early stage embryos, cord cells, cord blood, human induced pluripotent stem cells, bone marrow, undifferentiated stem cells, stem cells of mesoderm lineage, together with endothelial cells differentiated from stem cells. However, the present is not limited thereto.

[0113] According to another embodiment of the present disclosure, the antibody used for purifying a vascular endothelial cell may further include magnetic particles. Accordingly, the sorting step (S230) may include permeating a cell cluster to which an antibody to purify vascular endothelial cells is applied into a column receiving metallic particles therein, wherein magnetic force is externally applied to the column, and may include sorting cells bound to the metallic particles located inside the column as the vascular endothelial cells.

[0114] According to another feature of the present disclosure, the sorting (S230) may further include sorting the vascular endothelial cells by blocking the magnetic force applied from an outside of the column.

[0115] The vascular endothelial cells isolated, at the high purity, in this way are directly used for treatment of ischemic tissues, or may be used as a cell therapeutic agent for treatment of secondary induced diseases such as cardiovascular vessel diseases such as ischemic heart disease, arteriosclerosis, myocardial infarction and angina pectoris.

Example 1: Detection of Vascular Endothelial Cells Using Antibodies to Purify Vascular Endothelial Cells According to Embodiment of Present Disclosure Antibody Against PECAM1

[0116] Hereinafter, referring to FIG. 3A and FIG. 3B, the evaluation results of the antibody for purifying the vascular endothelial cells according to an embodiment of the present disclosure will be described. In this connection, HUVEC cells, that is, human vascular endothelial cells were used as vascular endothelial cells. However, the effect of the antibodies to purify vascular endothelial cells in accordance with the present disclosure is not limited thereto. For example, the antibodies to purify vascular endothelial cells may have the same effect on a mouse vascular endothelial cell.

[0117] FIG. 3A shows the results of immunofluorescence staining analysis for each of the clones determined as multiple of positive clones reacting with PECAM1 in the preparation method of an antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 3B shows the results of immunofluorescence staining for antibodies obtained by the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0118] Referring to FIG. 3A, there are shown results of detection of vascular endothelial cells when using antibodies purified from 10 positive clones 3F10, 4F1, 4H2, 6A3, 6A5, 6B2, 6C6, 8E5, 8E9 and 8H10 as eventually determined as positive clones in the producing method of the antibody for purifying vascular endothelial cells according to an embodiment of the present disclosure. More specifically, the antibodies of 3F10, 4H2, 6A3, 6C6, 8E9 and 8H10 are specific to the extracellular matrix domain of PECAM1 present on the surface of vascular endothelial cells and thus detect the vascular endothelial cells in a strong signaling manner. In particular, the antibody of 8H10 detects PECAM1 with a stronger signal than other antibodies. Referring to FIG. 3B, there are shown results of detection of vascular endothelial cells when using the antibody of 8H10. The antibody of 8H10 may be obtained by producing antibodies from a mouse using the 8H10 clones that are highly reactive with vascular endothelial cells, and, then, by purifying the antibodies. In this connection, the concentration of the obtained antibody of 8H10 was 3.86 mg/ml. The content of the antibodies of 8H10 was diluted at a 1000:1 ratio to perform an immunofluorescence staining method for HUVEC cells. More specifically, the endothelial cells detected by the reaction of the antibody of 8H10 with the PECAM1 protein present on the cell surface were generally consistent with the vascular endothelial cells stained by DAPI (4′,6′-diamidine-2′-phenylindole dihydrochloride). That is, the antibody of the 8H10 may effectively detect the vascular endothelial cells and may be used for purifying vascular endothelial cells.

[0119] In particular, the antibody of 8H10 has specificity to vascular endothelial cells and has a high degree of binding to the vascular endothelial cells at a concentration of about 0.00386 mg/ml following the dilution at 1000:1.

[0120] From a result of Example 1, the antibody for purifying the vascular endothelial cells used in various embodiments of the present disclosure may include antibodies of 3F10, 4F1, 4H2, 6A3, 6A5, 6B2, 6C6, 8E5, 8E9 and 8H10, preferably, antibodies of 3F10, 4H2, 6A3, 6C6, 8E9 and 8H10, more preferably, the antibody of 8H10. However, the present invention is not limited thereto.

Example 2: Evaluation of Cell Specificity of Antibodies for Purifying Vascular Endothelial Cells According to Embodiment of Present Disclosure Antibody Against PECAM1

[0121] Hereinafter, referring to FIG. 4A and FIG. 4B, the results of evaluation of cell specificity of antibodies for purifying vascular endothelial cells according to an embodiment of the present disclosure will be described. In this connection, HUVEC cells as human vascular endothelial cells were used as vascular endothelial cells. As a negative control, fibroblasts were used. In this connection, the content of the antibodies in accordance with the present disclosure, that is, 3.86 mg/ml concentration of the antibodies of 8H10 as described above in Example 1 was diluted in each of ratios of 100:1, 200:1, 500:1 and 1000:1. Then, the diluted antibodies were applied to fibroblast cells and human vascular endothelial cells, respectively.

[0122] FIG. 4A and FIG. 4B show the results of evaluation of cell specificity using immunofluorescence staining method for antibodies obtained by the preparation method of the antibody against PECAM1 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0123] First, referring to (a), (b), (c) and (d) of FIG. 4A, the results of the application of the antibodies in accordance with the present disclosure as diluted in ratios of 100:1, 200:1, 500:1 and 1000:1 to the fibroblasts as the negative control are shown. More specifically, it is observed that fibroblasts emit blue light. However, as the fibroblasts have no specific binding to the antibody in accordance with the present disclosure, the antibody emitting green light is not observed.

[0124] In one example, referring to (a), (b), (c) and (d) of FIG. 4B, together with blue luminescent human vascular endothelial cells, a green luminescent antibody appears on the surface of the endothelial cell. In this connection, the antibody in accordance with the present disclosure specifically binds to PECAM1 on the surface of human vascular endothelial cells, allowing the detection of the human vascular endothelial cells. In particular, referring to (d) of FIG. 4B, the antibody in accordance with the present disclosure at a diluted state at 1000:1 allows a high level of detection of vascular endothelial cells. Thus, the antibody in accordance with the present disclosure has a high specificity to the vascular endothelial cells even at low concentrations. Even at low concentrations of the antibodies, the vascular endothelial cells may be detected efficiently. Furthermore, the antibody in accordance with the present disclosure may facilitate the acquisition of the endothelial cells at the high purity.

[0125] In particular, the antibody of 8H10 has the specificity to the vascular endothelial cells and has a high degree of binding to the vascular endothelial cells at a concentration of about 0.00386 mg/ml following a dilution at 1000:1.

[0126] From a result of Example 2, the antibody for purifying vascular endothelial cells, which are used in various embodiments of the present disclosure may be used to isolate high purity endothelial cells as the antibody binds specifically to the vascular endothelial cells.

[0127] Thus, the vascular endothelial cells isolated using the antibody for purifying the vascular endothelial cells in accordance with the present disclosure may have a high purity and thus may be stably clinically applied as an effective cell therapy for the prevention or treatment of cardiovascular vessel diseases.

[0128] More specifically, the antibodies used in the various embodiments of the present disclosure may purify, in a high purity, the vascular endothelial cells that may be applied directly to ischemic tissue. Thus, the antibodies may constitute a cell therapeutic composition for treatment of diseases requiring blood vessel generation, such as ischemic heart blood vessel disease, brain blood vessel disease, diabetic complications, and wound care.

Example 3: Detection of Vascular Endothelial Cells Using Antibodies to Purify Vascular Endothelial Cells According to Embodiment of Present Disclosure Antibody Against CDH5

[0129] Hereinafter, referring to FIG. 5A and FIG. 5B, the evaluation results of the antibody for purifying the vascular endothelial cells according to an embodiment of the present disclosure will be described. In this connection, HUVEC cells, that is, human vascular endothelial cells were used as vascular endothelial cells. However, the effect of the antibodies to purify vascular endothelial cells in accordance with the present disclosure is not limited thereto. For example, the antibodies to purify vascular endothelial cells may have the same effect on a mouse vascular endothelial cell.

[0130] FIG. 5A shows the results of immunofluorescence staining analysis for each of the clones determined as multiple of positive clones reacting with CDH5 in the preparation method of an antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure. FIG. 5B shows the results of evaluation using immunofluorescence staining for antibodies obtained by the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0131] Referring to FIG. 5A, there are shown results of detection of vascular endothelial cells when using antibodies purified from 10 clones 1A5, 3G9, 6A11, 6C9, 8D2, 9G1, 9G4, 9H10, 10D6 and 10E11 eventually determined as the positive clones in the producing method of the antibody for purifying vascular endothelial cells according to one embodiment of the present disclosure. More specifically, the antibodies of 3F10, 4H2, 6A3, 6C6, 8E9 and 8H10 are specific to the extracellular matrix domain of CDH5 present on the surface of vascular endothelial cells and thus detect the vascular endothelial cells in a strong signaling manner. In particular, the antibody of 10D6 detects CDH5 with a stronger signal than other antibodies.

[0132] Referring to FIG. 5B, there are shown results of detection of vascular endothelial cells using the antibody of 10D6. The antibody of 10D6 may be obtained by producing antibodies from a mouse using the 10D6 clones that are highly reactive with vascular endothelial cells, and, then, by purifying the antibodies. In this connection, the concentration of the obtained antibody of 10D6 was 3.86 mg/ml. The content of the antibodies of 10D6 was diluted at a 1000:1 ratio to perform an immunofluorescence staining method for HUVEC cells. More specifically, the endothelial cells detected by the reaction of the antibody of 10D6 with the CDH5 protein present on the cell surface were generally consistent with the vascular endothelial cells stained by DAPI (4′,6′-diamidine-2′-phenylindole dihydrochloride). That is, the antibody of the 10D6 may effectively detect the vascular endothelial cells and may be used for purifying vascular endothelial cells.

[0133] In particular, the antibody of 10D6 has specificity to vascular endothelial cells and has a high degree of binding to the vascular endothelial cells at a concentration of about 0.00386 mg/ml following the dilution at 1000:1.

[0134] From a result of Example 3, the antibody for purifying the vascular endothelial cells used in various embodiments of the present disclosure may include antibodies of 1A5, 3G9, 6A11, 6C9, 8D2, 9G1, 9G4, 9H10, 10D6 and 10E11, preferably, antibodies of 1A5, 6A11, 6C9, 9G1, 9G4, 9H10 and 10D6, more preferably, the antibody of 10D6. However, the present invention is not limited thereto.

Example 4: Evaluation of Cell Specificity of Antibodies for Purifying Vascular Endothelial Cells According to Example of Present Disclosure Antibody Against CDH5

[0135] Hereinafter, referring to FIG. 6A and FIG. 6B, the results of evaluation of cell specificity of antibodies for purifying vascular endothelial cells according to an embodiment of the present disclosure will be described. In this connection, as an experimental group, HUVEC cells as human vascular endothelial cells were used as vascular endothelial cells. As a negative control, fibroblasts were used. In this connection, the content of the antibodies in accordance with the present disclosure, that is, 3.86 mg/ml concentration of the antibodies of 10D6 as described above in Example 3 was diluted in each of ratios of 500:1, 1000:1, 1500:1 and 2000:1. Then, the diluted antibodies were applied to fibroblast cells and human vascular endothelial cells respectively.

[0136] FIG. 6A and FIG. 6B show the results of evaluation of cell specificity using immunofluorescence staining process for antibodies obtained by the preparation method of the antibody against CDH5 for purifying the vascular endothelial cells according to an embodiment of the present disclosure.

[0137] First, referring to (a), (b), (c) and (d) of FIG. 6A, the results of the application of the antibodies in accordance with the present disclosure as diluted in ratios of 500:1, 1000:1, 1500:1 and 2000:1 to the fibroblasts as the negative control are shown. More specifically, it is observed that fibroblasts emit blue light. However, as the fibroblasts has no specific binding to the antibody in accordance with the present disclosure, the antibody emitting green light is not observed.

[0138] In one example, referring to (a), (b), (c) and (d) of FIG. 6B, together with blue luminescent human vascular endothelial cells, a green luminescent antibody appears on the surface of the endothelial cell. In this connection, the antibody in accordance with the present disclosure specifically binds to CDH5 on the surface of human vascular endothelial cells, allowing the detection of the human vascular endothelial cells. In particular, referring to (d) of FIG. 6B, the antibody in accordance with the present disclosure at a diluted state at 2000:1 allows a high level of detection of vascular endothelial cells. Thus, the antibody in accordance with the present disclosure has a high specificity to the vascular endothelial cells even at low concentrations. Even at low concentrations of the antibodies, the vascular endothelial cells may be detected efficiently. Furthermore, the antibody in accordance with the present disclosure may facilitate the acquisition of the endothelial cells at the high purity.

[0139] In particular, the antibody of 10D6 has the specificity to the vascular endothelial cells and has a high degree of binding to the vascular endothelial cells at a concentration of about 0.00193 mg/ml following a dilution at 2000:1.

[0140] From a result of Example 4, the antibody for purifying vascular endothelial cells, which are used in various embodiments of the present disclosure may be used to isolate high purity endothelial cells as the antibody binds specifically to the vascular endothelial cells.

[0141] Thus, the vascular endothelial cells isolated using the antibody for purifying the vascular endothelial cells in accordance with the present disclosure may have a high purity and thus may be stably clinically applied as an effective cell therapy for the prevention or treatment of cardiovascular vessel diseases.

[0142] More specifically, the antibodies used in the various embodiments of the present disclosure may purify, in a high purity. the vascular endothelial cells that may be applied directly to ischemic tissue. Thus, the antibodies may constitute a cell therapeutic composition for treatment of diseases requiring blood vessel generation, such as ischemic heart blood vessel disease, brain blood vessel disease, diabetic complications, and wound care.

[0143] Although the embodiments of the present disclosure have been described in more detail with reference to the accompanying drawings, the present disclosure is not necessarily limited to these embodiments. Many changes may be made without departing from the spirit of the present disclosure. Accordingly, the embodiments as disclosed in the present disclosure are not intended to limit the technical spirit of the present disclosure, but to describe the present disclosure. However, the scope of the technical idea of the present disclosure is not limited to these embodiments. Therefore, it should be understood that the embodiments as described above are exemplary in all respects and not restrictive. The scope of protection of the present disclosure should be interpreted by the following claims. All technical ideas falling within the equivalent scope should be interpreted as being included in the scope of the present disclosure.

TABLE-US-00001 SEQUENCE LISTING FREE TEXT <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> ANTIBODY FOR PURIFYING VASCULAR ENDOTHELIAL CELL AND METHOD FOR MAKING THEREOF <130> 19PD5340PCT <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 574 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 1 Gln Glu Asn Ser Phe Thr Ile Asn Ser Val Asp Met Lys Ser Leu Pro Asp Trp Thr Val Gln Asn Gly Lys Asn Leu Thr Leu Gln Cys Phe Ala Asp Val Ser Thr Thr Ser His Val Lys Pro Gln His Gln Met Leu Phe Tyr Lys Asp Asp Val Leu Phe Tyr Asn Ile Ser Ser Met Lys Ser Thr Glu Ser Tyr Phe Ile Pro Glu Val Arg Ile Tyr Asp Ser Gly Thr Tyr Lys Cys Thr Val Ile Val Asn Asn Lys Glu Lys Thr Thr Ala Glu Tyr Gln Leu Leu Val Glu Gly Val Pro Ser Pro Arg Val Thr Leu Asp Lys Lys Glu Ala Ile Gln Gly Gly Ile Val Arg Val Asn Cys Ser Val Pro Glu Glu Lys Ala Pro Ile His Phe Thr Ile Glu Lys Leu Glu Leu Asn Glu Lys Met Val Lys Leu Lys Arg Glu Lys Asn Ser Arg Asp Gln Asn Phe Val Ile Leu Glu Phe Pro Val Glu Glu Gln Asp Arg Val Leu Ser Phe Arg Cys Gln Ala Arg Ile Ile Ser Gly Ile His Met Gln Thr Ser Glu Ser Thr Lys Ser Glu Leu Val Thr Val Thr Glu Ser Phe Ser Thr Pro Lys Phe His Ile Ser Pro Thr Gly Met Ile Met Glu Gly Ala Gln Leu His Ile Lys Cys Thr Ile Gln Val Thr His Leu Ala Gln Glu Phe Pro Glu Ile Ile Ile Gln Lys Asp Lys Ala Ile Val Ala His Asn Arg His Gly Asn Lys Ala Val Tyr Ser Val Met Ala Met Val Glu His Ser Gly Asn Tyr Thr Cys Lys Val Glu Ser Ser Arg Ile Ser Lys Val Ser Ser Ile Val Val Asn Ile Thr Glu Leu Phe Ser Lys Pro Glu Leu Glu Ser Ser Phe Thr His Leu Asp Gln Gly Glu Arg Leu Asn Leu Ser Cys Ser Ile Pro Gly Ala Pro Pro Ala Asn Phe Thr Ile Gln Lys Glu Asp Thr Ile Val Ser Gln Thr Gln Asp Phe Thr Lys Ile Ala Ser Lys Ser Asp Ser Gly Thr Tyr Ile Cys Thr Ala Gly Ile Asp Lys Val Val Lys Lys Ser Asn Thr Val Gln Ile Val Val Cys Glu Met Leu Ser Gln Pro Arg Ile Ser Tyr Asp Ala Gln Phe Glu Val Ile Lys Gly Gln Thr Ile Glu Val Arg Cys Glu Ser Ile Ser Gly Thr Leu Pro Ile Ser Tyr Gln Leu Leu Lys Thr Ser Lys Val Leu Glu Asn Ser Thr Lys Asn Ser Asn Asp Pro Ala Val Phe Lys Asp Asn Pro Thr Glu Asp Val Glu Tyr Gln Cys Val Ala Asp Asn Cys His Ser His Ala Lys Met Leu Ser Glu Val Leu Arg Val Lys Val Ile Ala Pro Val Asp Glu Val Gln Ile Ser Ile Leu Ser Ser Lys Val Val Glu Ser Gly Glu Asp Ile Val Leu Gln Cys Ala Val Asn Glu Gly Ser Gly Pro Ile Thr Tyr Lys Phe Tyr Arg Glu Lys Glu Gly Lys Pro Glu Tyr Gln Met Thr Ser Asn Ala Thr Gln Ala Phe Trp Thr Lys Gln Lys Ala Ser Lys Glu Gln Glu Gly Glu Tyr Tyr Cys Thr Ala Phe Asn Arg Ala Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys <210> 2 <211> 552 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 2 Asp Trp Ile Trp Asn Gln Met His Ile Asp Glu Glu Lys Asn Thr Ser Leu Pro His His Val Gly Lys Ile Lys Ser Ser Val Ser Arg Lys Asn Ala Lys Tyr Leu Leu Lys Gly Glu Tyr Val Gly Lys Val Phe Arg Val Asp Ala Glu Thr Gly Asp Val Phe Ala Ile Glu Arg Leu Asp Arg Glu Asn Ile Ser Glu Tyr His Leu Thr Ala Val Ile Val Asp Lys Asp Thr Gly Glu Asn Leu Glu Thr Pro Ser Ser Phe Thr Ile Lys Val His Asp Val Asn Asp Asn Trp Pro Val Phe Thr His Arg Leu Phe Asn Ala Ser Val Pro Glu Ser Ser Ala Val Gly Thr Ser Val Ile Ser Val Thr Ala Val Asp Ala Asp Asp Pro Thr Val Gly Asp His Ala Ser Val Met Tyr Gln Ile Leu Lys Gly Lys Glu Tyr Phe Ala Ile Asp Asn Ser Gly Arg Ile Ile Thr Ile Thr Lys Ser Leu Asp Arg Glu Lys Gln Ala Arg Tyr Glu Ile Val Val Glu Ala Arg Asp Ala Gln Gly Leu Arg Gly Asp Ser Gly Thr Ala Thr Val Leu Val Thr Leu Gln Asp Ile Asn Asp Asn Phe Pro Phe Phe Thr Gln Thr Lys Tyr Thr Phe Val Val Pro Glu Asp Thr Arg Val Gly Thr Ser Val Gly Ser Leu Phe Val Glu Asp Pro Asp Glu Pro Gln Asn Arg Met Thr Lys Tyr Ser Ile Leu Arg Gly Asp Tyr Gln Asp Ala Phe Thr Ile Glu Thr Asn Pro Ala His Asn Glu Gly Ile Ile Lys Pro Met Lys Pro Leu Asp Tyr Glu Tyr Ile Gln Gln Tyr Ser Phe Ile Val Glu Ala Thr Asp Pro Thr Ile Asp Leu Arg Tyr Met Ser Pro Pro Ala Gly Asn Arg Gln Gln Val Ile Ile Asn Ile Thr Asp Val Asp Glu Pro Pro Ile Phe Gln Gln Pro Phe Tyr His Phe Gln Leu Lys Glu Asn Gln Lys Lys Pro Leu Ile Gly Thr Val Leu Ala Met Asp Pro Asp Ala Ala Arg His Ser Ile Gly Tyr Ser Ile Arg Arg Thr Ser Asp Lys Gly Gln Phe Phe Arg Val Thr Lys Lys Gly Asp Ile Tyr Asn Glu Lys Glu Leu Asp Arg Glu Val Tyr Pro Trp Tyr Asn Leu Thr Val Glu Ala Lys Glu Leu Asp Ser Thr Gly Thr Pro Thr Gly Lys Glu Ser Ile Val Gln Val His Ile Glu Val Leu Asp Glu Asn Asp Asn Ala Pro Glu Phe Ala Lys Pro Tyr Gln Pro Lys Val Cys Glu Asn Ala Val His Gly Gln Leu Val Leu Gln Ile Ser Ala Ile Asp Lys Asp Ile Thr Pro Arg Asn Val Lys Phe Lys Phe Ile Leu Asn Thr Glu Asn Asn Phe Thr Leu Thr Asp Asn His Asp Asn Thr Ala Asn Ile Thr Val Lys Tyr Gly Gln Phe Asp Arg Glu His Thr Lys Val His Phe Leu Pro Val Val Ile Ser Asp Asn Gly Met Pro Ser Arg Thr Gly Thr Ser Thr Leu Thr Val Ala Val Cys Lys Cys Asn Glu Gln Gly Glu Phe Thr Phe Cys Glu Asp Met Ala Ala Gln Val Gly Val Ser Ile Gln

[0144] [National R&D projects that supported this invention]

[0145] [Assignment unique number] HI16C2211

[0146] [Department name] Ministry of Health and Welfare

[0147] [Research Management Professional Institution] Korea Health Industry Development Institute

[0148] [Research Project Name] Stem cells, regenerative medicine commercialization/performance-creating mediation research

[0149] [Research Study Name] Development of cell therapy for peripheral arterial disease using vascular endothelial cells differentiated from induced pluripotent stem cells [Contribution rate] 4/10

[0150] [Organizing Agency] Yonsei University Industry-Academic Cooperation Foundation

[0151] [Study period] 20161108˜20210731

[0152] [National R&D projects that supported this invention]

[0153] [Assignment unique number] HI15C2782

[0154] [Department name] Ministry of Health and Welfare

[0155] [Research Management Professional Institution] Korea Health Industry Development Institute

[0156] [Research Project Name] Development of advanced medical technology/stem cell, international cooperation in establishing a foundation for regenerative medicine

[0157] [Research Study Name] Development of direct cell conversion method to vascular endothelial cells using nanoparticles

[0158] [Contribution rate] 3/10

[0159] [Organizing Agency] Yonsei University Industry-Academic Cooperation Foundation

[0160] [Study period] 20151201˜20191130

[0161] [National R&D projects that supported this invention]

[0162] [Assignment unique number] 2015M3A9C6031514

[0163] [Department name] Ministry of Science and ICT

[0164] [Research Management Professional Institution] National Research Foundation of Korea

[0165] [Research Project Name] Biomedical technology development business

[0166] [Research Study Name] Differentiation and separation of induced pluripotent stem cells into subtypes of cardiomyocytes, and development of technology based on cell therapy and drug evaluation using the same

[0167] [Contribution rate] 3/10

[0168] [Organizing Agency] Yonsei University Industry-Academic Cooperation Foundation

[0169] [Study period] 20150601˜20200531