Anti-CD98 monoclonal antibody and use thereof

Abstract

The present invention relates to: monoclonal antibody NPB15, which superficially binds to CD98 on the surface of pure human pluripotent stem cells, quasi-human pluripotent stem cells, human cancer cells, and cancer stem cells; a hybridoma producing same; a composition for detecting undifferentiated pluripotent stem cells using same; a pharmaceutical composition for preventing / treating cancer using same; and a composition for diagnosing cancer using same.

Description

Anti-CD98 monoclonal antibodies and uses thereof

[0001] The present invention relates to a monoclonal antibody that specifically binds to human CD98, and more particularly, to a monoclonal antibody that recognizes CD98 expressed on the surface of human pluripotent stem cells and various cancer cells including liver cancer, a hybridoma that produces the monoclonal antibody, a composition for removing, separating, and analyzing undifferentiated pluripotent stem cells containing the monoclonal antibody, and a composition for anticancer treatment containing the monoclonal antibody.

[0002]

[0003] Human pluripotent stem cells (hPSCs) are cells with the ability to proliferate indefinitely and differentiate into all cells that make up the body. The first human pluripotent stem cells, human embryonic stem cells (hESCs), were established in 1998 from frozen human embryos by Dr. James Thomson's team in the United States. In 2007, human induced pluripotent stem cells (hiPSCs), similar to hESCs, were established through genetic introduction by Dr. Shinya Yamanaka's team in Japan. Professor Austin Smith of Cambridge University further classified mammalian pluripotent stem cells into two or three different states, and said that different pluripotent stem cells can be isolated from the pre-implantation epiblast or the post-implantation epiblast before and after the fertilized egg is implanted in the uterus. He named the pre-implantation pluripotent stem cells naive pluripotent and the post-implantation pluripotent cells primed pluripotent. Pure and semi-native pluripotent stem cells differ greatly in cell shape, growth rate, differentiation ability, methylation level, and germline transmission ability. The semi-native state grows in a largely flat state and has a slow growth rate, while the pure state grows in a small dome shape, grows well even when separated into single cells with trypsin, has a fast growth rate, is not yet biased in the developmental stage, is expected to have the ability to differentiate into more cells, has a lower methylation level, and is known as an early developmental pluripotent stem cell with excellent germline transmission ability. Semi-native human pluripotent stem cells can be induced into pure human pluripotent stem cells under various culture conditions.

[0004] The first idea that cancer can arise from stem cells was formally proposed in the early 19th century by Durante and Conheim as the "Embryonal Rest hypothesis" of cancer, which proposed that remnants of embryonic tissue remain in adult organs and develop into cancer. Molecules that are actively expressed during embryonic development, exist minimally in adult tissues, and are re-expressed when cancer occurs are called oncofetal antigens. Oncofetal antigens are currently important target molecules used in the diagnosis and treatment of cancer, and CA-125, CEA, AFP, cancer / testes antigen, POA, etc. are actually used as diagnostic markers in clinical practice. In addition, Cripto-1, Glypican-3, 5T4, M2A, etc. are known as oncofetal antigens, and antibodies targeting them are being developed as cancer therapeutic antibodies. However, ethical issues regarding human embryos have made oncofetal antigen research difficult. However, in 1998, with the establishment and cultivation of human embryonic stem cells, oncofetal antigens on the surface of human embryonic stem cells, such as 5T4, Cripto, and EpCAM, were discovered. Recent studies have confirmed the inhibitory effect against colon cancer, lung cancer, breast cancer, mesothelioma, and melanoma in mice injected with hPSCs such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). This proves that monoclonal antibodies that recognize oncofetal antigens present in hPSCs can actually inhibit the development of cancer cells expressing the same antigens. Therefore, research on oncofetal antigens in hPSCs can discover cancer stem cells or cancer cell-specific molecules, and target molecules for cancer therapy are being developed through this strategy.

[0005] CD98 (SLC3A2, 4F2) is a solute carrier family protein and a component of amino acid transporters. It functions by pairing with another solute carrier family protein, such as the L-type amino acid transporter 1 (LAT1 / SLC7A5) or the cystine / glutamate transporter (xCT / SLC7A11). CD98 was discovered by biochemically analyzing antigens bound by antibodies. CD98 is known to function as an amino acid transporter and transport amino acids into and out of cells. In particular, it pairs with LAT1 to transport branched chain amino acids (BCAA) and aromatic amino acids (AAA) into cells, playing an important role in the cell cycle and maintaining nucleotide pools, and a decrease in LAT1 is known to affect decreased cell proliferation. Another function is to pair with xCT to transport cystine into cells. Cystine is a material that synthesizes glutathione (GSH) within cells, and plays an important role in protecting cells by reducing intracellular reactive oxygen species and maintaining redox balance.

[0006] CD98, essential for cellular metabolism, is distributed evenly across most cells and tissues. However, recent studies have revealed that it is overexpressed in cancer cells, where it plays a crucial role in cell proliferation, carcinogenesis, and migration. CD98 overexpression is closely correlated with tumor progression and poor overall survival in various cancers, including lung and liver cancers. Recent studies have also shown that CD98 is particularly overexpressed in chemotherapy-resistant and radiotherapy-resistant cancer cells in various cancers. This suggests that CD98 is closely associated with cancer stemness and has been suggested as a cancer stem cell marker in head and neck cancer and oropharyngeal cancer. Therefore, CD98 is considered an attractive target for immunotherapy in various cancers. Indeed, a monoclonal antibody targeting CD98 and its humanized antibody (IGN523) have shown efficacy in the treatment of acute myeloid leukemia by blocking amino acid transport or inducing cell death through apoptosis.

[0007] Despite its superiority as an anticancer target, CD98 is distributed ubiquitously across various cells and tissues, including cancer. Therefore, antibody therapeutics that recognize CD98 on the surface of normal cells can induce various immune side effects. However, CD98 is a highly glycosylated protein, and highly glycosylated proteins exhibit structural differences due to differences in glycosylation levels between normal and cancer cells. Recently, active research has been conducted on antibodies that recognize glycosylation patterns and bind only to cancer-specific molecules. Similarly, differences in protein modifications, including glycosylation, between normal and cancer cells are likely to exist in CD98. Indeed, a monoclonal antibody has been reported that binds specifically to the high mannose-type N-glycan present on CD98 on the surface of myeloma cells from multiple myeloma patients, but not to normal leukocytes. This antibody showed significantly low binding affinity to normal tissues and specifically bound only to cancer cells, demonstrating anticancer effects through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The antibody against CD98, which was screened from a human antibody library, showed anticancer effects through ADCC and antibody-dependent cellular phagocytosis (ADCP) by macrophages in hepatoma, lymphoma, pancreatic cancer, and colon cancer in a mouse xenograft model. By redesigning it as a human antibody with higher affinity for CD98 in a low-pH cancer microenvironment, the side effects on normal tissues could be significantly reduced.In addition, CD98 was confirmed to be highly expressed on regulatory T cells (Treg), and it was revealed that the CD98 molecule on the surface of Treg interacts with CD147 of other Treg cells to maintain the expression of Foxp3 on Treg. Foxp3 plays a role in increasing the stability of Treg cells, helping them survive and function longer, and Treg cells suppress the host's immune function to promote cancer growth. Therefore, it is thought that the development of anticancer immunotherapy can also be done by inhibiting CD98 on the surface of Treg cells with antibodies.

[0008] We generated NPB15, an antibody that simultaneously binds to both pure human pluripotent stem cells and quasi-human pluripotent stem cells, from a monoclonal antibody pool generated by injecting pure human pluripotent stem cells into mice. The antigen recognized by NPB15 was significantly reduced when pluripotent stem cells were differentiated, demonstrating specificity for undifferentiated human pluripotent stem cells. NPB15 did not bind to normal cells such as peripheral blood mononuclear cells and hepatocytes, but bound well to human embryonic carcinoma, liver cancer, and lung cancer cells. Analysis of the NPB15 antigen revealed that it was CD98 (SLC3A2, 4F2). We hypothesized that NPB15, which recognizes CD98, could be developed as a novel antibody for cancer therapy, recognizing an oncofetal epitope simultaneously expressed in both human pluripotent stem cells and cancer cells. Evaluation of the function of CD98 in liver cancer and lung cancer cells revealed that CD98 promoted cancer cell growth, survival, and migration, and exhibited cancer stem cell characteristics. In addition, cancer cells that bound well to NPB15 showed higher growth and viability, and when NPB15 was administered to cancer cells, it showed a remarkable antibody-dependent cellular cytotoxicity effect (ADCC). In fact, when NPB15 was injected into a mouse xenograft model in which liver cancer cells were transplanted into nude mice, it showed an effective anticancer effect. Therefore, this invention was completed by confirming that NPB15 is an antibody that can be developed as an antibody for cancer treatment.

[0009]

[0010] The present invention aims to provide a monoclonal antibody that recognizes the CD98 protein expressed on the surface of human pure pluripotent stem cells, human quasi-pluripotent stem cells, cancer cells, and cancer stem cells.

[0011] The purpose of the present invention is to provide a composition for detecting undifferentiated pluripotent stem cells using an undifferentiated human pluripotent stem cell marker that utilizes the function of the monoclonal antibody.

[0012] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer and a composition for diagnosing cancer by utilizing the function of the monoclonal antibody.

[0013] The purpose of the present invention is to provide a hybridoma that produces the monoclonal antibody.

[0014]

[0015] 1. An antibody comprising a heavy chain variable region comprising a heavy chain complementary determining region 1 (HCDR1) consisting of an amino acid sequence of SEQ ID NO: 1, a HCDR2 consisting of an amino acid sequence of SEQ ID NO: 2, and a HCDR3 consisting of an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain complementary determining region 1 (LCDR1) consisting of an amino acid sequence of SEQ ID NO: 4, a LCDR2 consisting of an amino acid sequence of SEQ ID NO: 5, and a LCDR3 consisting of an amino acid sequence of SEQ ID NO: 6.

[0016] 2. An antibody comprising a heavy chain variable region consisting of an amino acid sequence of sequence number 7 and a light chain variable region consisting of an amino acid sequence of sequence number 8 in the above 1.

[0017] 3. In the above 1, the antibody is an antibody that binds to CD98 on the surface of human naive pluripotent stem cells, human prime pluripotent stem cells, human cancer cells, and cancer stem cells.

[0018] 4. An antibody according to 1 above, further comprising a heavy chain constant region derived from a human IgG1 constant region and a light chain constant region derived from a human kappa constant region.

[0019] 5. An antibody according to claim 1, comprising a heavy chain region consisting of an amino acid sequence of SEQ ID NO: 9 and a light chain region consisting of an amino acid sequence of SEQ ID NO: 10.

[0020] 6. A composition for detecting undifferentiated pluripotent stem cells comprising any one of the antibodies 1 to 5 above.

[0021] 7. A pharmaceutical composition for preventing or treating cancer comprising any one of the antibodies 1 to 5 above.

[0022] 8. A pharmaceutical composition for preventing or treating cancer, further comprising an anticancer substance conjugated to the antibody in the above 7.

[0023] 9. A pharmaceutical composition for preventing or treating cancer, wherein the anticancer substance in the above 8 is conjugated via a secondary antibody that binds to the antibody.

[0024] 10. A pharmaceutical composition for preventing or treating cancer, comprising at least one further selected from the group consisting of NK cells, macrophages, and neutrophils in the above 7.

[0025] 11. In the above 7, a pharmaceutical composition for preventing or treating cancer, wherein the cancer is cancer derived from cancer cells expressing CD98 protein on the cell membrane.

[0026] 12. In the above 7, the cancer is a pharmaceutical composition for preventing or treating cancer selected from the group consisting of liver cancer, lung cancer, embryonal carcinoma, pancreatic cancer, osteosarcoma, skin cancer, neuroblastoma, uterine cancer, prostate cancer, and colon cancer.

[0027] 13. Hybridoma producing the antibody of the above 1.

[0028] 14. A composition for diagnosing cancer comprising any one of the antibodies 1 to 5 above.

[0029] 15. A composition for diagnosing cancer, wherein the cancer in the above 14 is a cancer derived from cancer cells that express CD98 protein on the cell membrane.

[0030] 16. In the above 14, the cancer is a composition for diagnosing cancer selected from the group consisting of liver cancer, lung cancer, embryonal carcinoma, pancreatic cancer, osteosarcoma, skin cancer, neuroblastoma, uterine cancer, prostate cancer, and colon cancer.

[0031]

[0032] The monoclonal antibody of the present invention can recognize the CD98 protein expressed on the surface of human pure pluripotent stem cells, human quasi-pluripotent stem cells, cancer cells, and cancer stem cells. This antibody recognizes an undifferentiated human pluripotent stem cell marker and can be used in compositions for detecting undifferentiated pluripotent stem cells, etc. Furthermore, this antibody can be used in pharmaceutical compositions for preventing or treating cancer, compositions for diagnosing cancer, etc.

[0033]

[0034] Figure 1a shows the transformation of primed human pluripotent stem cells (primed H9) into small dome-shaped pure human pluripotent stem cells (naive H9 (2i / L / F / A)) in 2i / L / F / A medium.

[0035] Figure 1b is a diagram showing the expression of primed human pluripotent stem cell markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD24 and naive human pluripotent stem cell markers CD7, CD77, CD130 in primed H9 and naive H9 (2i / LF / A) cells using flow cytometry (solid lines represent each monoclonal antibody, and shaded background includes only the secondary antibody).

[0036] Figure 1c is a picture of NPB15 antibody purified through affinity chromatography using Protein G agarose beads from NPB15 hybridoma cell culture. The left side shows the result after electrophoresis and staining with CBB R-250 solution, and the right side shows the result after Western blotting and the NPB15 antibody was confirmed using mIgG-(H+L)-HRP secondary antibody. Here, HC stands for immunoglobulin heavy chain, and LC stands for immunoglobulin light chain.

[0037] Figure 2 shows the human pluripotent stem cells, 2i / L / F / A, pure human pluripotent stem cells induced in 2i / L / X / F / P medium, mouse embryonic fibroblasts (MEFs), 10 -5 This figure shows the expression level of NPB15 in H9 cells treated with M retinoic acid (RA) for 7 days, as confirmed by flow cytometry.

[0038] Figure 3 is a drawing showing the expression level of NPB15 in human embryonic carcinoma cells (NT2 / D1, 2102Ep), hepatoma cells (Huh7, HepG2, SNU387, SNU449, PLC / PRF / 5, SK-Hep1), non-small cell lung cancer cells (NCI-H460, A549), normal hepatocytes, and peripheral blood mononuclear cells (PBMC) confirmed by flow cytometry.

[0039] Figure 4a is a picture showing NT-2 / D1, 2102Ep, and A549 cells whose cell surfaces were labeled with biotin, immunoprecipitated with NPB15, developed on an SDS-PAGE gel, transferred to a nitrocellulose membrane, and then the size of the NPB15 antigen was confirmed with streptavidin-horse radish peroxidase (SA-HRP).

[0040] Figure 4b is a diagram showing the size of the target antigen for LC-MS / MS analysis after immunoprecipitation of A549 cells with NPB15, CBB staining, and subsequent analysis.

[0041] Figure 4c is a drawing showing the portion of the peptide sequence obtained as a result of LC-MS / MS that matches the amino acid sequence of Lymphocyte activation antigen (CD98).

[0042] Figure 5a is a picture showing the size of the NPB15 antigen confirmed by SA-HRP after immunoprecipitation of biotin-labeled A549 cells with NPB15, expansion on SDS-PAGE gel, and transfer to a nitrocellulose membrane.

[0043] Figure 5b is a picture showing that the same membrane as Figure 5a was subjected to Western blotting with a commercial anti-CD98 antibody, confirming that the antigen immunoprecipitated by NPB15 was recognized by the anti-CD98 antibody.

[0044] Figures 5c and 5d are pictures showing that after exogenous transfection of HEK293FT cells with a CD98-FLAG expression vector having a FLAG tag, immunoprecipitation was performed with control mouse IgG1, NPB15, anti-CD98, and anti-FLAG, respectively, and the antigen was confirmed at the same position with anti-CD98 and anti-FLAG, confirming that NPB15 directly recognizes and immunoprecipitates CD98. The arrow indicates CD98.

[0045] Figure 6a is a picture showing the decrease in CD98 expression confirmed through Western blotting after knocking down CD98 using CD98 siRNA (siCD98) in human liver cancer cell lines (SNU-449, Huh7 cells) and human lung cancer cell lines (NCI-H460).

[0046] Figure 6b is a picture confirming the decrease in CD98 expression on the cell surface in CD98 knockdown cancer cells using a flow cytometer. The isotype is a control isotype antibody, and the siCon is a control knockdown cell.

[0047] Figure 6c shows the cell counts measured to observe cancer cell growth in CD98-knocked down cancer cells (siCD98) and compared with the cell counts of the negative control group (siCon) (* p<0.05; ** p<0.01).

[0048] Figures 7a, 7b, and 7c show the results of measuring apoptosis by staining CD98 knockdown SNU-449, Huh7, and NCI-H460 cells with PI and Annexin V.

[0049] Figures 7d, 7e, and 7f are quantitative graphs representing the results of Figures 7a to 7c, showing that apoptosis was increased in CD98 knockdown cancer cells (** p<0.01; *** p<0.001, ns, not significant).

[0050] Figure 8a is a picture of colonies formed by surviving a clonogenic survival assay using CD98 knockdown SNU-449, Huh7, and NCI-H460 cells, stained with crystal violet.

[0051] Figures 8b, 8c, and 8d are the results of statistical processing of the results of Figure 8a quantified using Image J (*** p<0.001).

[0052] Figure 9a shows the extent of scratch filling in a wound healing assay performed with CD98 knockdown (siCD98) SNU-449 cells compared to the negative control group (siCon) after 12 hours.

[0053] Figure 9b shows the scratch area quantified using Image J by repeating the process of Figure 9a, and then statistically processed and compared.

[0054] Figure 9c shows the extent of scratch filling in a wound healing assay performed with CD98 knockdown (siCD98) Huh7 cells compared to the negative control group (siCon) after 12 hours.

[0055] Figure 9d shows the results of repeating the process of Figure 9c, quantifying the scratch area with Image J, and comparing the results through statistical processing (* p<0.05; ** p<0.01; ns, not significant).

[0056] Figure 10a is a histogram showing the top 30% group with high antigen expression and the bottom 30% group with low antigen expression after labeling SNU-449 and NCI-H460 with NPB15 in a flow cytometry analysis.

[0057] Figure 10b shows the results of a clonogenic survival assay performed on cells from groups with high and low expression of NPB15, respectively, and staining the colonies with crystal violet.

[0058] Figures 10c and 10d are graphs showing that the number of colonies measured in Figure 10b was quantified and that colony survival rate increased in the CD98 upper group (*** p<0.001).

[0059] Figure 11a shows the results of flow cytometry analysis confirming changes in the expression of cancer stem cell marker CD44 in CD98 knockdown SNU-449 cells.

[0060] Figure 11b shows the results of flow cytometry analysis to confirm changes in CD44 expression in CD98 knockdown NCI-H460 cells.

[0061] Figure 11c is a quantification of the results of Figure 11a (*** p<0.001).

[0062] Figure 11d is a quantification of the results of Figure 11b (** p<0.01; *** p<0.001).

[0063] Figure 11e shows the results of comparing the activity of ALDH, a cancer stem cell marker, in CD98 knockdown SNU-449, Huh7, and NCI-H460 cells (** p<0.01).

[0064] Figure 11f is a picture showing that CD98 expression was reduced by knocking down CD98 using CD98 siRNA (siCD98) in human hepatoma cell line Huh7 cells, and that EpCAM, a cancer stem cell marker, was reduced in the same cells by Western blotting.

[0065] Figure 11g is the statistical result of repeating the experimental results of Figure 11f (** p<0.01; *** p<0.001; ns, not significant).

[0066] Figure 12a is a picture of liver cancer cells Huh7 cultured into tumorspheres with cancer stem cell characteristics.

[0067] Figure 12b compares the increased expression of NPB15 and cancer stem cell markers CD133 and EpCAM in adherent Huh7 cells and tumorigenic Huh7 cells using flow cytometry.

[0068] Figure 12c quantifies the results of Figure 12b (* p<0.05; ** p<0.01).

[0069] Figure 13a shows the results of amplifying the NPB15 heavy chain by PCR, confirmed on an agarose gel.

[0070] Figure 13b shows the results of PCR amplification of the light chain of NPB15.

[0071] Figure 13c shows the results of cutting the pBluescript KS+ vector with EcoRI and SalI restriction enzymes to clone the NPB15 heavy chain.

[0072] Figure 13d shows the results of cutting the pBluescript KS+ vector with HindIII and SalI restriction enzymes to clone the NPB15 light chain.

[0073] Figure 14 shows the results of cloning and sequence analysis of the variable region heavy chain portion of NPB15, expressed in DNA and amino acid sequences. The portion corresponding to the CDR is indicated in bold.

[0074] Figure 15 shows the results of cloning and sequence analysis of the variable region light chain portion of NPB15, expressed in DNA and amino acid sequences. The portion corresponding to the CDR is indicated in bold.

[0075] Figure 16a shows the results of agarose gel analysis confirming that the heavy chain variable region of NPB15 and the signal peptide for secretion were linked through PCR.

[0076] Figure 16b shows the results of cutting the pdCMV-dhfr Vector with restriction enzymes EcoRI and ApaI to confirm cloning of the heavy chain variable region portion (arrow) linked to the signal sequence into the vector.

[0077] Figure 16c shows the results of agarose gel analysis confirming that the light chain variable region and signal peptide of NPB15 were linked through PCR.

[0078] Figure 16d shows the results of cutting the pdCMV-dhfr Vector with restriction enzymes HindIII and BsiWI to confirm cloning of the light chain variable region portion (arrow) linked to the signal sequence into the vector.

[0079] Figure 17 shows the heavy chain amino acid sequence (SEQ ID NO: 9) of a chimeric antibody that links the heavy chain variable region of the NPB15 antibody and the human IgG1 constant region.

[0080] Figure 18 shows the light chain amino acid sequence (SEQ ID NO: 10) of a chimeric antibody that links the light chain variable region of the NPB15 antibody and the human kappa constant region.

[0081] Figure 19a shows the results of purifying the chimeric antibody from the cell culture medium using protein G-agrarose after introducing the chimeric NPB15 antibody expression vector into HEK293FT and staining it with Coomassie brilliant blue (CBB) on an SDA-PAGE gel. Human IgG was used as a control.

[0082] Figure 19b shows the results of transferring Figure 19a to a nitrocellulose membrane and performing Western blotting with an anti-human IgG-HRP antibody to verify that it is a human antibody.

[0083] Figure 19c is a diagram showing that NPB15 and Chi-NPB15 bind to liver cancer cells in the same manner through flow cytometry analysis on the liver cancer cell line Huh7.

[0084] Figure 20 is a drawing showing the results of using an ADCC reporter assay kit (Promega) to observe whether the chimeric antibody NPB15 can bind to liver cancer cells and induce ADCC signals. Human IgG was serially diluted with a control antibody and chimeric NPB15 and combined with Huh7 cells, and the ADCC signal was amplified by co-culturing Jurkat cells and measuring the emitted light to measure whether the chimeric antibody NPB15 induces ADCC signals (* p <0.05; *** p value <0.001).

[0085] Figure 21a is a schematic diagram showing the experimental process for observing the anticancer effect of NPB15 in a nude mouse model in which Huh7 cells, which are liver cancer cells, were xenografted.

[0086] Figure 21b shows the change in tumor volume observed in the control group injected with mIgG1 isotype control antibody and the experimental group injected with NPB15 in nude mice xenografted with Huh7 cells, which are liver cancer cells (*** p value <0.001).

[0087] Figure 21c shows the results comparing the sizes of tumor masses harvested from mice sacrificed 20 days after antibody administration. The sizes of tumor masses were compared between the control group injected with mIgG1 isotype control antibody and the experimental group injected with NPB15.

[0088] Figure 21d compares the weight of tumors in the control group administered mIgG1 isotype control antibody and the experimental group administered NPB15. The size of tumors was compared through 2-way ANOVA, and the weight of tumors was compared through Student's t-test (* p value <0.05).

[0089] Figure 21e shows the results of measuring the change in body weight of the control group administered mIgG1 isotype control antibody and the experimental group administered NPB15 after antibody administration to determine whether there were any side effects during antibody injection.

[0090] Figure 22a is a schematic diagram showing the experimental process for observing the anticancer effect of Chi-NPB15 in a nude mouse model in which Huh7 cells, a liver cancer cell line, were xenografted.

[0091] Figure 22b shows the change in tumor volume observed in the control group injected with hIgG1 isotype control antibody and the experimental group injected with Chi-NPB15 in nude mice xenografted with Huh7 cells, which are liver cancer cells (*** p value <0.001).

[0092] Figure 22c shows the results of measuring the change in body weight of the control group administered hIgG1 isotype control antibody and the experimental group administered Chi-NPB15 after antibody administration to determine whether there were any side effects during antibody injection.

[0093]

[0094] The present invention will be described in detail below. Unless otherwise defined, all terms used in this specification have the same general meaning as those skilled in the art to which the present invention pertains. If there is a conflict between the meaning of a term used in this specification and the meaning used in this specification, the meaning used in this specification shall prevail.

[0095] The present invention relates to an antibody comprising a heavy chain variable region comprising a heavy chain complementary determining region 1 (HCDR1) comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3; and a light chain variable region comprising a light chain complementary determining region 1 (LCDR1) comprising SEQ ID NO: 4, an LCDR2 comprising SEQ ID NO: 5, and an LCDR3 comprising SEQ ID NO: 6.

[0096] The monoclonal antibody of the present invention can bind to the CD98 protein of pure human pluripotent stem cells, quasi-human pluripotent stem cells, cancer cells, and cancer stem cells.

[0097] In the present invention, the naive human pluripotent stem cell refers to a pre-implantation human pluripotent stem cell, which is said to have pluripotency in a naive or ground state. The post-implantation human pluripotent stem cell refers to a primed human pluripotent stem cell with pluripotency in a primed state. As shown in Fig. 1a, the naive human pluripotent stem cell is distinguished from the primed pluripotent stem cell, which grows in a form similar to a flat colony, in that individual cells separate and grow in a small dome shape. When the naive human pluripotent stem cell is further divided, it can be divided into the ground state stem cell, and the intermediate, formative, or naive-like state pure pluripotency stem cell.

[0098] The above cancer cells are cancer cells that express CD98 on the cell surface, and are, without limitation, cancer cells of the present invention. Examples thereof include liver cancer, lung cancer, human embryonal carcinoma, embryonal carcinoma, pancreatic cancer, osteosarcoma, skin cancer, neuroblastoma, uterine cancer, prostate cancer, kidney cancer, breast cancer, colon cancer, stomach cancer, biliary tract cancer, bladder cancer, ovarian cancer, and brain tumor. In one embodiment of the present invention, when two embryonal carcinoma cell lines (NT2 / D1, 2102Ep), two lung cancer cell lines (A549, NCI-H460), and six liver cancer cell lines (SNU-449, PLC / PRF / 5, HepG2, Huh7, SNU-387, SK-Hep1) were treated with the antibody of the present invention and flow cytometry analysis was performed, it was confirmed that the antibody of the present invention strongly bound to the cell lines. However, the present invention is not limited thereto.

[0099] The cancer stem cells described above are, without limitation, any cancer stem cell that expresses CD98 on its cell surface. For example, they may be liver cancer stem cells. In one embodiment of the present invention, it was confirmed that the antibody of the present invention specifically recognizes liver cancer cells with cancer stem cell characteristics. However, this is not a limitation.

[0100] The sequences of the heavy chain variable region and light chain variable region of the antibody of the present invention are not limited as long as it has the HCDR sequence of SEQ ID NO: 1 to 3 and the LCDR sequence of SEQ ID NO: 4 to 6. For example, it may have a heavy chain variable region sequence including SEQ ID NO: 7 and a light chain variable region sequence including SEQ ID NO: 8, but is not limited thereto.

[0101] The above sequence number 7 refers to the heavy chain variable region sequence shown in Fig. 13, and the above sequence number 8 refers to the light chain variable region sequence shown in Fig. 14.

[0102] Sequence numbers 1 to 8 are as shown in Table 1.

[0103] Sequence numberAmino acid sequence1Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr2Thr Ile Ser Asp Gly Gly Ser Tyr Ala Tyr Tyr Pro Asp Asn Met Lys Gly3Asp Arg Ala Tyr Tyr Tyr Gly Ser Ser Tyr Gly Phe Ala Tyr4Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Asn Thr Phe Leu His5Lys Val Ser Asn Arg Phe Ser6Ser Gln Ser Thr His Val Pro Tyr Thr7Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Asp Gly Gly Ser Tyr Ala Tyr Tyr Pro Asp Asn Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Asn Leu Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp Arg Ala Tyr Tyr Tyr Gly Ser Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala8Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Asn Thr Phe Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys LeuLeu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

[0104]

[0105] The present invention provides an antibody further comprising a heavy chain constant region derived from a human IgG1 constant region and a light chain constant region derived from a human kappa constant region.

[0106] The present invention provides an antibody comprising a heavy chain region comprising an amino acid sequence of SEQ ID NO: 9 and a light chain region comprising an amino acid sequence of SEQ ID NO: 10.

[0107] The present invention provides a mouse-human chimeric antibody (Chi-NPB15).

[0108] In the present invention, the antibody may specifically mean a monoclonal antibody, and in the present invention, the monoclonal antibody means a protein molecule that is directed against a single antigenic site (a single epitope) and specifically binds thereto. For the purpose of the present invention, the monoclonal antibody of the present invention specifically binds to CD98, a cell surface molecule of pure human pluripotent stem cells, quasi-human pluripotent stem cells, and cancer cells, and is therefore a protein molecule that recognizes cell surface molecules of pure human pluripotent stem cells, quasi-human pluripotent stem cells, and cancer cells.

[0109] The main part of an antibody that recognizes a specific epitope of an antigen and forms an antigen-antibody complex is the variable region of the heavy and light chains, particularly the complementarity determining region (CDR), which contributes to the formation of this complex. Therefore, the present invention includes chimeric antibodies, humanized antibodies, etc., including the variable region of the monoclonal antibody of the present invention, particularly the CDR, within the scope of the present invention. For example, in one embodiment of the present invention, a mouse-human chimeric antibody (Chi-NPB15) may be mentioned. In addition, as long as the variable region of the monoclonal antibody of the present invention, particularly the CDR, is the same, the IgG subclasses such as IgG1 to IgG4 are also included within the scope of the present invention. In addition, the present invention includes functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains, as long as they have the binding characteristics as described above. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

[0110] The present invention relates to a composition for detecting undifferentiated pluripotent stem cells comprising the above antibody.

[0111] The above undifferentiated pluripotent stem cells are undifferentiated human pluripotent stem cells. The undifferentiated pluripotent stem cells may be pure human pluripotent stem cells or quasi-human pluripotent stem cells. The pure human pluripotent stem cells and quasi-human pluripotent stem cells are as described above.

[0112] The above detection can identify or isolate undifferentiated pluripotent stem cells. Since the antibody recognizes a specific marker of undifferentiated pluripotent stem cells, undifferentiated stem cells can be isolated or removed using a composition containing the antibody. The detection composition can also be expressed as a composition for removing undifferentiated pluripotent stem cells, a composition for isolating undifferentiated pluripotent stem cells, or a composition for analyzing undifferentiated pluripotent stem cells.

[0113] The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the antibody.

[0114] The composition can be used for the prevention or treatment of cancer, regardless of whether the cancer is caused by cancer cells expressing CD98 on the cell surface. Examples thereof include liver cancer, lung cancer, embryonal carcinoma, pancreatic cancer, osteosarcoma, skin cancer, human embryonal cancer, neuroblastoma, uterine cancer, prostate cancer, colon cancer, stomach cancer, biliary tract cancer, kidney cancer, bladder cancer, ovarian cancer, and brain tumor. In one embodiment of the present invention, it was confirmed that the composition of the present invention had an anticancer effect when treated with liver cancer cells SNU-449 and Huh7 and lung cancer cells NCI-H460. However, the present invention is not limited thereto.

[0115] The composition can be used for the prevention or treatment of cancer, regardless of whether the cancer is caused by cancer stem cells expressing CD98 on their cell surface. Cancers caused by cancer stem cells frequently relapse after treatment and often exhibit anticancer drug resistance. The antibody of the present invention can specifically recognize CD98 on the surface of cancer stem cells, and thus the composition comprising the antibody of the present invention has the effect of inhibiting cancer recurrence, making it effective for the prevention or treatment of cancer.

[0116] The above cancer prevention and treatment may be mediated by the antibody-dependent cellular cytotoxicity (ADCC) mechanism. ADCC is mediated by immune cells expressing the Fc gamma receptor (FcγR) when antibodies bind to tumor or viral antigens. These cells recognize the antibody-bound cells and initiate an immune response to eliminate the antigen. For example, macrophages and neutrophils recognize the Fc region and eliminate antigen-expressing cells through phagocytosis, while natural killer cells (NK cells) recognize the Fc region and secrete granzymes to eliminate antigen-expressing cells. As mentioned above, the pharmaceutical composition of the present invention can specifically bind to cancer cells expressing CD98, and when an antibody manufactured in the form of IgG1 is included in the composition and treated, CD98-positive cancer cells bound to the antibody are killed through the ADCC mechanism, thereby exhibiting a preventive and therapeutic effect on cancer.

[0117] The above cancer prevention and treatment may be based on immune checkpoint inhibitor therapy (ICI therapy). Cancer cells evade immune cell-mediated cancer cell elimination mechanisms through mechanisms such as suppression of immune function, T cell immunotolerance, or immune-editing. ICI is one such evasion mechanism, which expresses proteins on the cell surface that interfere with cancer cell destruction, thereby activating inhibitory immune checkpoints and evading immune cell attacks. ICI therapy is a treatment method based on the principle that antibodies that bind to proteins that activate inhibitory immune checkpoints inhibit the function of the proteins, thereby activating immune cell-mediated cancer cell death mechanisms. As mentioned above, the pharmaceutical composition of the present invention can specifically bind to CD98, which performs the function of an inhibitory immune checkpoint, and therefore, when treated with IgG4 containing the CDR of the antibody, it can induce cancer cell death by antibody NK cells, macrophages, and neutrophils.

[0118] In the present invention, a pharmaceutical composition for preventing or treating cancer containing the antibody may be a composition in which the antibody and an anticancer substance are conjugated, and the conjugation may be accomplished by a linker sequence or a secondary antibody binding to the antibody.

[0119] In the present invention, the secondary antibody refers to an antibody that specifically binds to the amino acid sequence of another antibody (primary antibody) that binds to an antigen, and corresponds to a term that is different from the primary antibody that directly binds to the target antigen in terms of the target object. In the present invention, the linker sequence is used to connect an anticancer substance and an antibody. For example, the linker sequence can be attached to the Fc sequence of the antibody, and the linker sequence and the anticancer substance can be connected to produce an antibody-linker-anticancer substance (antibody-linker-drug) form and used. The linker or the secondary antibody are both used for the purpose of connecting the antibody of the present invention with the anticancer substance, and can be used by a method used by those skilled in the art regardless of the secondary antibody sequence or linker sequence if the purpose is to conjugate the antibody and the drug.

[0120] In the present invention, the anticancer agent may be used without limitation as long as it is an anticancer agent known in the art, for example, erlotinib (TARCEVA(TM), Genentech / OSI Pharm.), bortezomib (VELCADE(TM), Millenium Pharm.), fulvestrant (FASLODEX(TM), Astrazeneca), sutent (SUl 1248, Pfizer), letrozole (FEMARA(TM), Novartis), imatinib mesylate (GLEEVEC(TM), Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin(TM), Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE(TM), Wyeth), lapatinib (GSK572016, GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs.), and gefitinib (IRESSA(TM), Astrazeneca), AG 1478, AG 1571 (SU 5271; Sugen), thiotepa, and CYTOXAN(TM) cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic organism topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spogistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; Antibiotics such as enedine antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omega1I (Angew Chem Intl. Ed. Engl. (1994) 33:183-186); dynemicins including dynemicin A; bisphosphonates such as clodronate; esperamicins; and neocarzinostatin chromophore and related chromoprotein enedine antibiotic chromophores), aclacinomycins, actinomycins, anthramycins, azaserine, bleomycins, cactinomycins, carabicins, carminomycins, carzinophilin, clomomycinis, dactinomycins, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN(TM) doxorubicin (morpholino-doxorubicin, (including cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as proline; aceglatone; aldophosphamide glycoside; aminoebulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defopamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidadinine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; Pirarubicin; losoxantrone; podopiphyllinic acid; 2-ethylhydrazide; procarbazine; PSK(TM) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazoic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verraculin A, roridin Ala, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL(TM) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANETM cremophor-free, albumin-engineered nanoparticle preparation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE(TM) docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR(TM) Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE(TM) vinorelbine;Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); a retinoid such as leninoic acid; capecitabine; and any pharmaceutically acceptable salt, acid or derivative thereof.

[0121] When using a pharmaceutical composition by conjugating an anticancer agent, cancer prevention or treatment can be induced through the mechanism of action of the antibody-drug conjugate (ADC). The ADC is a method of conjugating a drug to an antibody and injecting it. This method induces the internalization of the antibody in cells expressing an antigen that specifically binds to the antibody, thereby delivering the drug into the cell, thereby achieving cell-specific drug delivery.

[0122] The pharmaceutical composition of the present invention is formulated into a unit dosage form suitable for administration into a patient's body according to a conventional method in the pharmaceutical field, preferably into a form of a formulation useful for administration of a protein drug, and can be administered by a parenteral route including, but not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal, or rectal routes using an administration method conventionally used in the art.

[0123] Suitable dosage forms for this purpose include various oral preparations such as tablets, pills, dragees, powders, capsules, syrups, solutions, gels, suspensions, emulsions, microemulsions, etc., and parenteral preparations such as injections, infusions, and sprays such as hyposprays, etc. In the case of preparations for injection or infusion, they may take the form of suspensions, solutions, or emulsions, etc., and may contain formulating agents such as suspending agents, preservatives, stabilizers, and / or dispersing agents. In addition, the antibody molecules may be formulated in a dry form that can be used by readjusting them with a suitable sterile liquid before use.

[0124] The antibody as an active ingredient of the pharmaceutical composition of the present invention can be administered to mammals, including humans, at a dose of 0.01 to 50 mg / kg body weight, preferably 0.1 to 20 mg / kg body weight, once or in several divided doses per day. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors, such as the disease to be prevented or treated, the severity of the disease, the route of administration, the patient's body weight, age and sex, drug combination, reaction sensitivity and resistance / response to treatment, and therefore, the dosage does not limit the scope of the present invention in any way.

[0125] The present invention provides a method for preventing, diagnosing or treating cancer, comprising administering to a subject an antibody of the present invention.

[0126] The present invention provides a method for preventing, diagnosing or treating cancer, comprising administering to a subject a pharmaceutical composition of the present invention.

[0127] Administration refers to introducing the antibody or pharmaceutical composition of the present invention into a subject using an appropriate method and in an appropriate dosage. The antibody or pharmaceutical composition of the present invention may be administered via various oral or parenteral routes as long as it can reach the target tissue. Specifically, the antibody or pharmaceutical composition may be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalational, intraocular, or intradermal routes.

[0128] An individual refers to an animal that has developed or is likely to develop cancer, or any other animal for which information for diagnosing cancer is sought. The animal may be a mammal, preferably a human, but is not limited thereto.

[0129] In addition, the present invention relates to a hybridoma producing the above antibody.

[0130] In the present invention, hybridoma means a hybridoma cell or hybridoma cell line, which is a cell created by artificially fusing two different types of cells, and means a cell or cell line in which two or more homologous or heterologous cells are fused using a substance that causes cell fusion, such as polyethylene glycol (PEG) or a virus of some kind, thereby integrating different functions of each cell into a single cell. In one embodiment of the present invention, the present invention provides a hybridoma that produces a monoclonal antibody NPB15. Specifically, the hybridoma of the present invention comprises 2 × 10 6Three days prior, H9-quasi-human pluripotent stem cells were injected into the right footpad of a mouse, and then the same number of H9-quasi-human pluripotent stem cells and pure human pluripotent stem cells were injected into the right and left footpads of the mouse seven times at three-day intervals, respectively. Lymphocytes were isolated from the left groin of the mouse and fused with myeloma cancer cells to produce hybridomas. Hybridomas that secrete monoclonal antibodies can be cultured in large quantities in vitro or in vivo.

[0131] The monoclonal antibody produced by the above hybridoma may be used without purification, but to obtain the best results, it is preferable to purify it to a high purity (e.g., 95% or higher) using a method well known in the art to which the present invention pertains. Such purification techniques include, for example, gel electrophoresis, dialysis, salt precipitation, and chromatography, through which it can be separated from the culture medium or ascites fluid. In one embodiment of the present invention, the culture medium of the hybridoma was purified using protein G-Sepharose column chromatography for mass production of the monoclonal antibody. However, any method known in the art for culturing and purifying the hybridoma is possible and is not limited thereto.

[0132] The present invention also relates to a cancer diagnostic composition comprising the antibody. As described above, the antibody can specifically bind to the CD98 protein of cancer cells. Since the CD98 protein is generally overexpressed in various cancer cells, if the CD98 antigen is identified in a target cell, it can be said that the cell is highly likely to be a cancer cell. Accordingly, when a composition comprising the antibody is applied to a cancer cell expressing CD98, the antibody binds to the CD98 antigen of the cancer cell to form an antigen-antibody complex, thereby allowing the cancer cell to be classified into cancer cells that bind to the antibody and normal cells that do not bind to the antibody.

[0133] The cancers that can be diagnosed by the above cancer diagnostic composition are preferably cancers known to overexpress CD98, such as liver cancer, lung cancer, embryonal carcinoma, pancreatic cancer, osteosarcoma, skin cancer, human embryonal cancer, neuroblastoma, uterine cancer, prostate cancer, colon cancer, stomach cancer, biliary tract cancer, kidney cancer, bladder cancer, ovarian cancer, brain tumor, etc., but are not limited thereto.

[0134]

[0135] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0136]

[0137] Example 1: Culturing human pluripotent stem cells and establishing pure human pluripotent stem cells.

[0138] 1-1. Cultivation of human quasi-pluripotent stem cells

[0139] Primed human pluripotent stem cells (Primed hPSCs) H9 were cultured according to a previously described method according to the protocol provided by Wicell Research Institute (Korean Patent Nos. 10-2344948 and 10-2235935). First, mouse embryonic fibroblasts (MEFs) from pregnant CF1 mice were isolated and cultured, and then treated with γ-irradiation or mitomycin (10 μg / ml) to serve as feeder cells. They were cultured in DMEM / F12 (Invitrogen) medium supplemented with 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, and 8–12 ng / ml bFGF. When subcultured every 5–6 days, they were treated with collagenase IV (1 mg / ml) for 5 minutes, cut into appropriate sizes with a yellow tip, and cultured on new feeder cells. As a result, the appearance of cultured quasi-human pluripotent stem cells that grew large and flat can be seen (Fig. 1a).

[0140]

[0141] 1-2. Establishment of pure human pluripotent stem cells

[0142] Hereinafter, the term “naive hPSC” refers to stem cells that express markers of naive hPSCs, unlike primed hPSCs, and refers to stem cells that express markers of stem cells in the ground state or an intermediate state such as intermediate or formative. Primed hPSCs H9 were used to induce conversion into naive hPSCs and cultured, and the specific protocol followed a previously known method (Korean Patent No. 10-2344948). First, primed hPSCs H9 were treated with collagenase IV (1 mg / ml) for 5 minutes, cut into an appropriate size using a yellow tip, and then transferred onto pre-disposed MEFs. The following day, the cells were cultured in 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% KOSR, KO-DMEM, 1% L-glutamine, 1% NEAA, 0.1 mM β-mearcaptoethanol, 1x penisillin / streptomycin) (Duggal et al. Stem Cells 33:2686, 2015). Within 3 to 5 days of culture, they transformed into dome-shaped cells, and were subcultured at 3 to 4-day intervals using 0.05% (v / v) trypsin-EDTA or accutase (StemPro). To enhance cell survival, 10 μM Y-27632 (Tocris) was added only during the first passage. As a result, pure human pluripotent stem cells grew in a small, dome-shaped pattern, compared to the flat, cultured quasi-human pluripotent stem cells (Fig. 1a). Both quasi- and pure human pluripotent stem cells, cultured for at least 4–5 passages, were used for immunological experiments.

[0143]

[0144] 1-3. Cell surface antigen staining by flow cytometry

[0145] To compare cultured primed H9 and naive H9 human pluripotent stem cells, the expression of SSEA3, SSEA4, TRA-1-60, TRA-1-81, and CD24, known as human primed pluripotent stem cell markers, was first analyzed using flow cytometry. For flow cytometry, cells separated with collagenase IV were dissociated into single cells using TrypLE (Invitrogen), passed through a 40 μm strainer, and then approximately 2–3 × 10 per sample were collected. 5 Cells were used. After reacting with SSEA3, SSEA4, TRA-1-60, TRA-1-81, and CD24-PE antibodies at 4°C for 30 minutes, the cells were washed twice with PBA (1% (w / v) bovine serum albumin, 0.02% (w / v) NaN3 in PBS), and then reacted with the primary antibody corresponding to anti-rat IgM-FITC, anti-mouse IgM-FITC, or anti-mouse IgG-FITC (BD Biosciences) for another 30 minutes at 4°C. After washing twice with PBA, antibody binding was analyzed for PI (propidium iodide)-negative cells using FACSCalibur and Cell Quest software (BD sciences). In addition, the expression of CD7, CD77, and CD130, known as background pure human pluripotent stem cell markers, was observed in the same way. As a result, it was observed that among the markers of semi-human pluripotent stem cells, the expression of SSEA3, SSEA4, TRA-1-60, TRA-1-81, and CD24 decreased in pure human pluripotent stem cells, and among the markers of pure human pluripotent stem cells known as CD7, CD77, and CD130, CD77 and CD130 increased in naive H9, confirming that pure human pluripotent stem cells were induced in 2i / L / F / A medium (Fig. 1b).

[0146]

[0147] Example 2. Production of NPB15 hybridoma

[0148] 2-1. Immunotherapy for the production of antibodies specific to pure human pluripotent stem cells

[0149] To produce a monoclonal antibody against pure human pluripotent stem cells, pluripotent stem cells were injected into the footpads of mice, and the specific protocol followed a previously well-described method (Korean Patent No. 10-2344948). First, 2 × 10 pluripotent stem cells were injected into the right footpads of 11 6-week-old female Balb / c mice. 6 H9 cells, which are semi-human pluripotent stem cells, were injected at -3, 0, 3, 6, 13, 17, 20, and 21 days, and the same number of pure human pluripotent stem cells, H9 cells, were injected into the left footpad at 0, 3, 6, 13, 17, 20, and 21 days, and on the 22nd day, the immunized mice were subjected to cervical dislocation, and the popliteal lymph nodes were removed from the left hind paws, fat and muscle tissues were removed, and the popliteal lymph nodes were ground using frosted slide glass with an uneven surface and prepared as single cells. Left lymph node cells and FO myeloma cells were mixed at a ratio of 1:5, washed twice with serum-free medium DMEM (Invitrogen), and then 1 ml of 50% (w / v) PEG1500 (polyethylene glycol, BMS, Seoul, Korea) was added at a rate of 25 drops per minute. After 1 minute, 3 ml of DMEM medium was added successively 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. 2 × 10 cells were seeded in a 96-well plate. 5 Cells / wells were cultured for approximately 2 weeks in DMEM medium containing 20% ​​(v / v) FBS, 1xHAT (Sigma-Aldrich), and HCS (hybridoma cloning supplement), and only hybridomas producing antibodies were selected using a sandwich ELISA method.

[0150]

[0151] 2-2. Cloning and purification of NPB15 hybridoma

[0152] To select antibody-expressing clones, a sandwich ELISA (Sandwich Enzyme Linked Immunosorbent Assay) method was used. 100 μl of hybridoma culture was added to a plate coated with 2 μg / ml of anti-mouse IgG or IgM antibody as a capture antibody, and the plate was incubated at 37°C for 1 hour. The plate was then incubated with a 1 / 5,000 dilution of anti-mouse IgG-HRP or anti-mouse IgM-HRP (horseradish peroxidase, Sigma-Aldrich) for another hour. After washing three times with PBST (0.05% (v / v) Tween-20 in PBS), a substrate solution containing OPD (o-phenylenediamine, Sigma) and H2O2 was added, and the absorbance was measured at 492 nm to first select antibody-producing clones. Next, the culture medium of each clone was first selected for hybridomas that bind to pure human pluripotent stem cells using the same method as the above FACS analysis, and among these, 33 hybridomas that bind to both semi-human pluripotent stem cells and pure human pluripotent stem cells were additionally selected, and stable clones were secured by repeating subcloning, and among these, the antibody NPB15 is an antibody with IgG1 and Kappa chains, and the antibody was purified for the next experiment in this invention.

[0153] Antibody purification was performed using NPB15 hybridoma cell culture medium using the Protein G agarose bead method. To this end, the collected hybridoma cell culture medium was placed in a column filled with Protein G agarose beads (Amicogen, Korea) to allow the antibody to bind to the Protein G agarose beads. After washing the beads with PBS (pH 8.0), the antibody was eluted from the Protein G agarose beads by adding 10 ml of 0.1 M glycine (pH 2.8) and 1 M Tris-HCl (pH 9.0). The purity of the purified NPB15 antibody was confirmed through SDS-PAGE and CBB R250 staining (Fig. 1c, left). The purified antibody was further analyzed by Western blotting. When anti-mouse antibody (goat-a-mIgG-(H+L)-HRP) was used as a secondary antibody, the heavy chain (HC) and light chain (LC) of the IgG chain were detected, confirming that NPB15 was well purified (Fig. 1c, right). The purified NPB15 antibody was then used to analyze the recognition antigen and its characteristics.

[0154] As in the examples 1-3 above, flow cytometry using NPB15 antibody showed that NPB15 simultaneously bound to pure human pluripotent stem cells and pseudo-human pluripotent stem cells derived from 2i / L / F / A (Korean Patent No. 10-2344948) and 2i / L / X / F / P medium (Korean Patent No. 10-2235935), but did not bind to mouse embryonic fibroblasts cultured together (Fig. 2). In addition, pseudo-human pluripotent stem cells were cultured at 10 -5When NPB15 binding was analyzed after inducing differentiation by treating with M retinoic acid (all-trans-retinoic acid, Sigma-Aldrich) for 7 days, a significant decrease was observed, indicating that NPB15 antigen is a molecule specific to undifferentiated human pluripotent stem cells that is expressed specifically in pluripotent stem cells and decreases when differentiation is induced (Fig. 2).

[0155]

[0156] Example 3. Analysis of the binding specificity of NPB15 to various cancer cells and normal cells.

[0157] 3-1. Culturing of various cancer cells and isolation of blood cells

[0158] Human embryonic carcinoma cells NT-2 / D1 and 2102Ep were cultured in DMEM medium supplemented with 10% FBS, 1% (v / v) NEAA, 100 U / ml penicillin-G, and 100 μg / ml streptomycin. Hepatoma and lung cancer cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). Human peripheral blood mononuclear cells (PBMC) were isolated using the Ficoll-Paque Plus method (GE Healthcare, Seoul, Korea). Human primary hepatocytes were purchased from Thermo Fisher Scientific (Waltham, USA) and cultured according to the supplier's protocol. Flow cytometry was performed to observe the binding of monoclonal antibody NPB15 to various cells. Various cancer cells were detached with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea) and neutralized with cell culture medium containing 10% fetal bovine serum (VWR). Then, the cells were passed through a 40 μm strainer (SPL, Pocheon, Korea) to prepare single cells. Approximately 1 × 10 cells per ml were used. 6After mixing each sample with PBA (1% bovine serum albumin, 0.02% NaN3 in PBS), the antibody NPB15 was reacted at 4℃ for 30 minutes, and the primary antibody corresponding to anti-mouse IgG-FITC (Invitrogen) was reacted for another 20 minutes at 4℃. After washing twice with PBA, the antibody reactivity was analyzed for PI (propidium iodide)-negative cells using FACSCalibur and Cell Quest software (BD sciences). NPB15 bound well to two embryonal carcinoma cell lines (NT-2 / D1, 2102Ep), two lung cancer cell lines (A549, NCI-H460), and six hepatoma cell lines (SNU-449, PLC / PRF / 5, HepG2, Huh7, SNU-387, SK-Hep1). However, it did not bind to normal human peripheral blood mononuclear cells (PBMCs) or normal hepatocytes (Fig. 3 and Table 2). Table 2 below analyzes the binding affinity of NPB15 to various cells.

[0159] These results indicate that the antigen epitope recognized by the NPB15 antibody is not expressed on the surface of human normal cells such as PBMC and hepatocytes, but is well expressed on the surface of human pluripotent stem cells, embryonic carcinoma cells, lung cancer cells, and liver cancer cells.

[0160] Cell lineCell originReactivity by flowcytometryPrimed H9Human embryonic stem cell, primed state+++Naive H9Human embryonic stem cell, naive state+++RA-H9Retinoic acid-treated primed H9+MEFMuse embryonic fibroblast-NT2 / D1Human embryonic carcinoma cell+E embryonic carcinoma cell+++A549Human non-small cell carcinoma+++NCI-H460Human large cell lung carcinoma+++SNU-387Human hepatocellular carcinoma+++SNU-449Human hepatocellular carcinoma+++HepG2Human hepatocellular carcinoma++++HepG2Human hepatocellular carcinoma++++HepG2 carcinoma+++PLC / PRF / 5Human hepatocellular carcinoma+++SK-Hep1Human hepatic adenocarcinoma+++HepatocyteHuman primary hepatocyte-PBMCHuman peripheral blood monocellular cell-Binding affinity is defined as: +++ Strong, + Weak, - Absent

[0161]

[0162] 실시예 4. NPB15가 인식하는 항원 동정

[0163] 4-1. NPB15가 인식하는 세포표면 단백질 분자의 확인

[0164] Biotin labeling (biotinylation) of the surface of human embryonic carcinoma cell lines NT-2 / D1, 2102Ep, and human lung cancer cell line A549 was performed with a slight modification of the protocol suggested by EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific). Human embryonic carcinoma NT-2 / D1 cells cultured in 100-millimeter cell culture plates were washed twice with PBS (pH 7.4), and 5 ml of cold PBS (pH 8.0) containing 0.5 mg of biotin was added, followed by incubation at 4°C for 30 minutes. After washing three times with cold PBS (pH 8.0), biotin-labeled cells were incubated with lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 μg / ml Aprotinin, 100 μg / ml PMSF (phenylmethylsulphonyl fluoride), 5 μg / ml Leupeptin, 1 mM NaF, 1 mM Na3VO4) at 4°C for 30 min, centrifuged at 12,000 rpm for 40 min to remove nuclei, and stored at -70°C until use. To remove proteins that nonspecifically bind to Protein G agarose (Amicogen, Jinju, Korea), approximately 2.0 × 10 720 μl of Protein G agarose was added to the cell lysate of canine cells and reacted at 4°C for 2 hours. The Protein G agarose was recovered by centrifugation. The sample washed 5 times in lysis buffer was used as a negative control (Con). In order to immunoprecipitate the antigen recognized by NPB15, 20 μl of Protein G agarose was first added to the cell lysate and reacted for another 4 hours at 4°C. Proteins nonspecifically bound to Protein G agarose were first removed by centrifugation. In order to immunoprecipitate the antigen specifically recognized by the monoclonal antibody NPB15, 4 μg of NPB15 antibody was added to the supernatant of the recovered cell lysate and reacted at 4°C for 12 hours, and then 20 μl of Protein G agarose was added and reacted for another 4 hours at 4°C. The immunoprecipitated immune mixture by centrifugation was washed five times in lysis buffer, and 5X sample buffer was added to elute the antigen bound to the antibody, and boiled at 100℃ for 10 minutes. The negative control protein (Con) and the eluted protein were separated by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Western blotting was performed on a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder for 1 hour at room temperature. After washing three times with 0.1% TBST (Tris-Buffered Saline, 0.1% Tween), streptavidin-HRP (SA-HRP, GE healthcare) was reacted for 1 hour at room temperature. After washing three times with 0.1% TBST, the biotin-labeled protein was confirmed using an ECL detection kit (Advansta). As a result, it was confirmed that NPB15 could immunoprecipitate proteins of approximately 120 kDa and 90 kDa from cell lysates of human embryonal carcinoma cells NT-2 / D1 and lung cancer cell line A549 (Fig. 4a).And in another human embryonic carcinoma cell line, 2102Ep, it was confirmed that a protein of approximately 120 kDa was immunoprecipitated by NPB15 (Fig. 4a).

[0165]

[0166] 4-2. Identification of antigens immunoprecipitated by NPB15

[0167] A polyacrylamide gel containing proteins immunoprecipitated from A549 cell lysate by monoclonal antibody NPB15 was stained with PageBlue Protein Staining Solution (Thermo Fisher Scientific) according to the supplier's protocol (Fig. 4b). The protein band stained at the 120-kDa position, presumed to be a protein immunoprecipitated by monoclonal antibody NPB15, was excised from the gel and subjected to LC-MS / MS (Liquid Chromatography with Tandem Mass Spectrometry) analysis (ProteomeTech, Seoul, Korea). The UniProt database (https: / / www.uniprot.org / ) was used for protein identification from the mass spectrum after analysis. As a result, the amino acid sequence of the protein was confirmed to be partially identical to that of SLC3A2 (CD98hc 4F2) (Fig. 4c; SEQ ID NO: 11). The underlined amino acids represent the amino acid sequence actually confirmed through mass spectrometry, and it can be seen that 59 amino acids are identical to CD98.

[0168]

[0169] 4-3. Verification of the identified NPB15 antigen

[0170] To confirm whether the antigen protein of NPB15 is CD98, immunoprecipitation, Western blotting, and gene knockdown analysis by RNA interference were performed using commercially available antibodies, gene expression vectors, and siRNA against CD98. First, to confirm whether the protein immunoprecipitated by NPB15 was CD98, a rabbit anti-CD98 monoclonal antibody (Cell signal technology) was purchased and used for immunoprecipitation and Western blotting. A549 cell lysate whose cell surface was labeled with biotin was prepared using the method described in Example 4-1, and immunoprecipitation was performed with NPB15 in the same manner as described above. Afterwards, the eluted protein and the negative control protein (Con) without the antibody were separated through 8% SDS-PAGE and transferred to a nitrocellulose membrane for Western blotting. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour. After washing three times with 0.1% TBST, rabbit anti-CD98 monoclonal antibody (α-CD98) was added and reacted at 4℃ for 12 hours. After washing three times with 0.1% TBST, anti-rabbit IgG-HRP (1:7,000; Bethyl Laboratories) was further reacted at room temperature for 1 hour. After washing three times with 0.1% TBST, the result was confirmed using an ECL detection kit (GE healthcare). As a result, it was confirmed that the 90 kDa and 120 kDa proteins immunoprecipitated by NPB15 were recognized by the rabbit anti-CD98 monoclonal antibody, confirming that NPB15 binds to and immunoprecipitates CD98 (Fig. 5a). To confirm that the detected protein is the cell surface membrane protein recognized by NPB15, the same nitrocellulose membrane was placed in a buffer containing SDS and β-mercaptoethanol and reacted at 50°C for 30 minutes to remove the detection signal, then streptavidin-HRP (SA-HRP) was added and reacted at room temperature for 1 hour. 0.After washing three times with 1% TBST, the biotin-labeled proteins were detected using an ECL detection kit. As a result, immunoprecipitates of the monoclonal antibody NPB15 were detected again at 90 and 120 kDa (Fig. 5b). These results indicate that the antigen immunoprecipitated from the A549 cell surface by NPB15 is CD98, which corresponds to approximately 90 and 120 kDa.

[0171] To distinguish whether NPB15 directly recognizes and immunoprecipitates CD98 or indirectly recognizes and precipitates it, the CD98 gene expression vector pCMV3-SLC3A2-Flag (Sino Biological) was purchased and Flag-tagged recombinant CD98 was overexpressed in human embryonic kidney cells HEK293FT. The cell lysates were subjected to immunoprecipitation and Western blotting using mouse anti-Flag monoclonal antibody (Invitrogen) and rabbit anti-CD98 monoclonal antibody (α-CD98). To overexpress CD98 in HEK293FT, 3.5 × 10 cells per well were seeded in 6-well cell culture plates. 5HEK293FT cells were cultured for 24 hours, and 2 μg of the known pCMV3-CD98-Flag vector DNA was mixed with 6 μg of PEI (Polyetherimide), and the mixture was reacted at room temperature for 20 minutes. The mixture was added to the cell culture medium to perform transfection. After culturing the cells for an additional 48 hours, a cell lysate was prepared using the lysis method described in Example 4-1. The cell lysate was immunoprecipitated with the control group containing isotype mIgG1 (mIgG1), NPB15, anti-Flag antibody, and anti-CD98 antibody (α-CD98) as described above. Each immunoprecipitate was separated using 8% SDS-PAGE together with a negative control protein (Con) without antibody, and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour. After washing three times with 0.1% TBST, the cells were reacted with known mouse anti-Flag monoclonal antibodies or rabbit anti-CD98 monoclonal antibodies (α-CD98) at 4°C for 12 hours, respectively. After washing three times with 0.1% TBST, the cells were further reacted with anti-mouse IgG-HRP (1:10,000; Millipore) or anti-rabbit IgG-HRP (1:10,000) at room temperature for 1 hour. After washing three times with 0.1% TBST, the cells were confirmed using an ECL detection kit. As a result, it was observed that all proteins immunoprecipitated by NPB15, mouse anti-Flag monoclonal antibodies, and rabbit anti-CD98 antibodies (α-CD98) were recognized by known anti-CD98 antibodies (α-CD98) (Fig. 5c) and anti-Flag antibodies (α-Flag) (Fig. 5d). This result verifies that the monoclonal antibody NPB15 of the present invention is an antibody that directly recognizes the CD98 protein present on the surface of the human lung cancer cell line A549.

[0172]

[0173] Example 5. Functional analysis of CD98 antigen

[0174] 5-1. CD98 knockdown and cell proliferation analysis in liver cancer cell lines

[0175] To analyze how CD98, an antigen of NPB15, affects the migration, survival, and growth of liver cancer cells, gene knockdown was performed in liver cancer cell lines using siRNA targeting the CD98 gene. siRNA against CD98 (Bioneer, Daejeon, Korea) was purchased and transfected into liver cancer cell lines SNU-449 and Huh7 and lung cancer cell lines NCI-H460. 50 nM of negative control siRNA (siCon; Genolution, Seoul, Korea), siRNA against CD98 (siCD98), and Lipofectamine RNAiMAX (Invitrogen) were each diluted in TOM (Welgene, Gyeongsan, Korea) and incubated at room temperature for 5 minutes. Afterwards, each siRNA dilution and RNAiMAX dilution were mixed and incubated at room temperature for 20 minutes. 1.5 × 10 per well in a 6-well plate. 5 - 3.0X10 5The cancer cell lines were spread and siRNA-RNAiMAX dilutions were added and cultured for 24 hours. After 24 hours, the second transfection was repeated in the same way and cultured for an additional 48 hours. Seventy-two hours after the first transfection, the cells were detached with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea), neutralized with cell culture medium containing 10% fetal bovine serum (VWR), lysed with the cell lysis buffer of Example 4-1, and then boiled at 100°C for 10 minutes with 5X sample buffer. The siCon protein and siCD98 protein were separated by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then subjected to Western blotting with a nitrocellulose membrane. As a result, it can be seen that the CD98 protein was reduced by approximately 99.9%, 72%, and 80%, respectively, in the liver cancer cells SNU-449, Huh7, and lung cancer cell line NCI-H460 knocked down with siRNA compared to α-tubulin used as a negative control, indicating that the knockdown was effective (Fig. 6a). Next, the expression of cell surface CD98 in the knocked down cancer cells was analyzed by flow cytometry using NPB15 as in Example 3. As a result, the expression of cell surface CD98 was significantly reduced in all cancer cells, indicating the result of CD98 knockdown (Fig. 6b). To analyze cancer cell proliferation in CD98-knocked-down liver cancer cells SNU-449, Huh7 and lung cancer cells NCI-H460, the knocked-down cancer cells were cultured for 48 h as described above, detached with 0.05% Trypsin-EDTA (Welgene), neutralized with cell culture medium containing 10% fetal bovine serum (VWR), and recovered by centrifugation at 100 × g for 5 min. The cells were suspended in 1 ml of PBS and then mixed with 0.1% PBS at a 1:1 ratio.After mixing with a 4% Trypan Blue solution and incubating at room temperature for 3 minutes, cells were placed on a hemocytometer and counted under a microscope, with only unstained, viable cells counted. As a result, a decrease in cell number was observed when CD98 was knocked down in all cancer cells (Figure 6c). This result suggests that CD98 is required for cancer cell proliferation.

[0176]

[0177] 5-2. Measurement of cancer cell proliferation and apoptosis in CD98 knockdown cancer cell lines

[0178] To analyze apoptosis in CD98-knocked down liver cancer cells SNU-449, Huh7 and lung cancer cells NCI-H460, annexin V assay was performed on the knocked down cells. To analyze apoptosis, cancer cell lines SNU-449, Huh7, and NCI-H460 were dissociated into single cells using 0.05% Trypsin (Welgene), neutralized with cell culture medium containing 10% fetal bovine serum (VWR), and passed through a 40 μm strainer. Approximately 2–3 x 10 per sample 5Cells were used. After washing twice with PBS (pH 7.4) to remove residual substances, the dead cells were stained by reacting with propidium iodide and annexin V-FITC at room temperature for 15 minutes. As a result, it was confirmed that the number of cells positive for both annexin V and PI increased by about 6%, from about 15% to 31% in CD98 knockdown SNU-449 (Figs. 7a and 7d), and in CD98 knockdown Huh7, the number of cells positive for both annexin V and PI did not differ significantly, but the number of annexin V-positive cells increased from about 10% to 46%, from about 36% (Figs. 7b and 7e). In CD98 knockdown NCI-H460, annexin-positive cells were approximately 15% higher than in the negative control group, but both annexinⅤ and PI-positive cells significantly increased by approximately 24% (from 20% to 44%) in CD98 knockdown, indicating that apoptosis was further increased in CD98 knockdown (Figures 7c and 7f). These results demonstrate that cancer cells with reduced CD98 undergo more active apoptosis, suggesting that CD98 is a key molecule in preventing cell death.

[0179]

[0180] 5-3. Clonogenic survival analysis of cancer cells in CD98 knockdown cancer cell lines

[0181] To investigate the role of CD98 in the survival and proliferation of liver and lung cancer cells, a clonogenic survival assay was performed using the CD98 knockdown SNU-449, Huh7, and NCI-H460 cells prepared in Example 5-1. The CD98 knockdown SNU-449, Huh7, and NCI-H460 cells prepared in Example 5-1 were detached with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea), neutralized with cell culture medium containing 10% fetal bovine serum (VWR), and passed through a 40 μm strainer (SPL, Pocheon, Korea) to prepare single cells. 1.0 × 10 per well in a 6-well plate. 4 The cells were spread out one by one to allow them to disperse and attach as single cells, and cultured for 10 days at 37℃, 5% CO2. The formed colonies were stained with crystal violet solution, and the total number of colonies was analyzed using Image J program. The same experiment was repeated three times to statistically represent the colony forming ability of the cells (Fig. 8a). CD98 knockdown reduced the number and size of SNU-449, Huh7, and NCI-H460 colonies by approximately 70%, 97%, and 61%, respectively (Figs. 8b to 8d). These results demonstrate that CD98 is an important cell surface molecule that enhances the survival and promotes proliferation of hepatoma cells under low-density seeding stress.

[0182]

[0183] 5-4. Analysis of cancer cell migration in CD98 knockdown cancer cell lines

[0184] To investigate the role of CD98 in the essential migratory ability of cancer cells in metastasis, a wound healing assay was performed using the CD98 knockdown SNU-449 and Huh7 cells described above. The CD98 knockdown SNU-449 and Huh7 cells prepared in Example 5-1 described above were seeded at 1.5 × 10 per well in a 24-well plate.5 Cells were seeded and incubated overnight until they filled 90% of the culture dish. The day after seeding, the cell monolayer was scratched using a yellow pipette tip to create a scratch wound. The culture medium was aspirated to remove cell debris, washed twice with PBS (pH 7.4), and the medium was replaced with 5% FBS and incubated at 37°C for 24 h. The scratches were photographed at 0 and 12 h after scratching, and the scratch area was quantified using Image J. After 12 h, the scratch area of ​​the negative control group (siCon) was reduced by approximately 34%, whereas that of CD98-knockdown SNU-449 cells (siCD98) was reduced by approximately 8% (Figures 9a and 9b). Similarly, in Huh7 cells, the scratch area of ​​the negative control group (siCon) was reduced by approximately 24% after 12 hours, whereas that of CD98-knockdown Huh7 cells (siCD98) was reduced by approximately 6% (Figures 9c and 9d). These results demonstrate that CD98 is a key cell surface molecule that promotes cancer cell migration, resulting from the decreased migration ability of cancer cells due to CD98 deficiency.

[0185]

[0186] Example 6. Comparative analysis of CD98-high / low cancer cell separation and cancer cell clonogenic survival ability by NPB15.

[0187] To investigate the effect of CD98 expression on cell growth, human liver cancer cells SNU-449 and lung cancer cells NCI-H460 were sorted using monoclonal antibody NPB15 into the top 30% cells with high CD98 expression (CD98-high) and the bottom 30% cells with low CD98 expression (CD98-low), and then clonogenic survival assays were performed for comparison of each cell group. For cancer cell sorting, 3.0 × 10 7The cells were detached with 0.05% Tryspsin-EDTA and neutralized with cell culture medium containing 10% fetal bovine serum. The cells were passed through a 40 μm strainer to prepare single cells. After washing twice with PBS (pH 7.4) to remove residual substances, they were allowed to bind to NPB15 using the method of Example 3. Afterwards, the upper 30% and lower 30% cells expressing CD98 were separated using a cell sorter (BD bioscience, BD FACSAriaⅢ) (Fig. 10a). The separated cells were seeded into 6-well plates at a density of 1.0 × 10 per well using the same method as Example 5-3. 4 The cells were spread out one by one to allow them to disperse and attach as single cells and cultured for 10 days at 37℃, 5% CO2 conditions. To analyze the size and number of colonies formed after 10 days, they were stained with crystal violet solution (Fig. 10b). The stained cell colonies were analyzed using the Image J program, and the same experiment was repeated three times and quantified (Figs. 10c and 10d). In the liver cancer cell line SNU-449, the clonogenic viability was confirmed to be reduced by approximately 35% in the CD98-low group compared to the CD98-high group (Fig. 10c). In the lung cancer cell line NCI-H460, the clonogenic viability was reduced by approximately 39% in the CD98-low group compared to the CD98-high group (Fig. 10d). These results suggest that the higher the CD98 expression, the higher the cancer cell survival and cancer stemness, and that the NPB15-high cancer cells by the NPB15 of the present invention are cells with higher cancer survival and cancer stemness than the NPB15-low cancer cells.

[0188]

[0189] Example 7. Analysis of cancer stem cell potential of NPB15-positive cancer cells

[0190] 7-1. Analysis of cancer stem cell marker expression in CD98 knockdown cancer cell lines

[0191] Cancer stem cells are stem cells that provide cancer recurrence or resistance to chemotherapy / radiotherapy, and CD98 has already been reported as a cancer stem cell marker in head and neck cancer. To determine whether CD98 is also associated with cancer stem cell markers in liver or lung cancer, we observed changes in the expression of representative cancer stem cell markers such as CD44, ALDH, EpCAM, and CD13 in CD98 knockdown cancer cells. As described in Example 5, CD98 was knocked down in SNU-449, Huh7, and NCI-H460 using siRNA (siCD98), and the expression of CD44, a representative cancer stem cell marker, was first analyzed by flow cytometry in the knockdown cell lines. To verify that CD98 was knocked out, we first confirmed that the expression level of CD98 was reduced in siCD98-transfected SNU-449 and NCI-H460 cells by comparing cells transfected with siCon using NPB15 and cells transfected with siCD98 (Figs. 11a and 11b). Next, the expression of CD44 in human lung cancer and liver cancer was analyzed by flow cytometry after binding with mouse anti-CD44 (α-CD44, Biolegend) antibody for 30 minutes at 4°C, followed by reaction with anti-mouse-IgG-FITC antibody as a secondary antibody for 30 minutes at 4°C. The analysis results showed that the CD44 expression level in the liver cancer cell line SNU-449 was reduced by approximately 49% (Figs. 11a and 11c). In the lung cancer cell line NCI-H460, a decrease in CD44 expression was confirmed by approximately 22% (Fig. 11b and 11d). This suggests that CD98 can be used as a marker of cancer stem cells in liver and lung cancer. In addition, to investigate the correlation with cancer stem cell markers, the activity change of ALDH, another cancer stem cell marker, was measured in SNU-449, Huh7, and NCI-H460. The activity of ALDH was measured by incubating 1 × 10 CD98 knockdown cancer cells transfected with negative control siRNA (siCon) and siCD98, respectively.6 After preparing each cell, we purchased an ALDH activity assay kit from Abcam and followed the supplier's protocol. The microplates that showed discoloration due to ALDH activity were measured at OD450 nm using an absorbance meter. As a result, approximately 19, 18, and 22% decreases in ALDH activity were observed in CD98 knockdown SNU-449, Huh7, and NCI-H460 cells, respectively (Fig. 11e). This suggests again that CD98 can be used as a marker of cancer stem cells in liver and lung cancer. The expression of EpCAM, another liver cancer stem cell marker, was analyzed by Western blotting using an anti-EpCAM antibody (AbCam) as in Example 4-1, and it was confirmed that approximately 60% of EpCAM expression was reduced in CD98 knockdown Huh7 cells (Figs. 11f and 11g). These results suggest that CD98 is an important molecule for maintaining cancer stemness in liver and lung cancer.

[0192]

[0193] 7-2. NPB15 binding analysis in tumorsphere culture

[0194] To generate tumorspheres with cancer stem cell characteristics using human hepatoma cell line Huh7, tumorsphere medium (50X B27, 20ng / ml EGF, 20ng / ml bFGF, DMEM / F12) was prepared. After straining through a 40μm cell strainer, 2.0X10 per well was seeded in a 24-well ultra-low attachment plate (Corning). 5Each well was inoculated and cultured in a 37°C incubator for 48 hours. After 48 hours, well-formed tumorspheres were observed (Fig. 12a). For subculture, all cells in each well were collected in a 50-ml tube and left at room temperature for 10 minutes until the cell pellet settled. After 10 minutes, the supernatant was carefully removed, 1 ml of medium (RPMI 1640, 10% FBS, 1% Antibiotic Antimycotic) was added, and the pellet was dispersed and collected in a 1.5-ml tube. The cells were washed with PBS (pH 7.4) and collected by centrifugation. PBS was removed from the cells, 1 ml of fresh medium was added, and 400 μl of Trypsin-EDTA was added and left at room temperature for 3 minutes. Prepare 9 ml of fresh media in a 15 ml tube, add the cell fluid in a 1.5 ml tube, mix, and centrifuge at 1500 rpm for 3 minutes. Inject 2.0 x 10 cells per well into a 24-well ultra-low attachment plate (Corning). 5 Each cell was inoculated and cultured for an additional 24 hours in a 37°C incubator. Similarly, adherent cells and tumor cells were detached using Trypsin-EDTA, and 1.5X10 each 5 After preparing each antibody, flow cytometry analysis was performed as described in Example 3-1. NPB15, CD133 (Abcam), a cancer stem cell marker, and EpCAM (Santa Cruz Biotechnologies) were used as primary antibodies, and the degree of binding of NPB15 to cancer stem cell markers in original adherent cells and tumor cells was compared using flow cytometry. As a result, it was observed that CD133 and EpCAM, which are cancer stem cell markers of liver cancer, increased in tumor cells, and the binding of NPB15 also significantly increased in tumor cells (Figures 12b and 12c). These results suggest that the NPB15 antibody is an antibody that specifically recognizes liver cancer cells with cancer stem cell characteristics.

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[0196] Example 8. Analysis of monoclonal antibody NPB15 gene and amino acid

[0197] 8-1. Cloning of the monoclonal antibody NPB15 gene

[0198] 5×10 rapidly growing hybridoma NPB15 cells 6After harvesting the dogs by centrifugation, they were washed with cold PBS and total RNA was extracted using RNAiso plus reagent (TaKaRa, Otsu, Japan) according to the supplier's protocol. The amount of RNA was quantified by measuring the A260 of the obtained total RNA. 1 unit of DNase I (TaKaRa) per 1 μg of total RNA was added and incubated at 37°C for 30 minutes to remove residual DNA. 1 μl of 50 mM EDTA was added and incubated at 85°C for 5 minutes to inactivate DNase I and denature total RNA. A reverse transcription-polymerase chain reaction (RT) mixture was prepared using total RNA and the Prime Script RT reagent Kit (TaKaRa), and cDNA was synthesized according to the supplier's protocol. For heavy chain cloning with synthesized cDNA, 25 pmoles of an oligonucleotide with the polymerase chain reaction primer 5'-gga gtc gac ATA GAC AGA TGG GGG TGT CGT TTT GGC-3' (SEQ ID NO: 12) corresponding to the IgG1 constant region, 25 pmoles of an oligonucleotide with the base sequence 5'MH1 5'-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TC-3' (SEQ ID NO: 13) corresponding to the N-terminus of the heavy chain antibody variable region, and 25 pmoles of an oligonucleotide 5'MH2 5'-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TCW GG-3' (SEQ ID NO: 14) corresponding to the heavy chain antibody variable region N-terminus were added to create a chain polymerization reaction mixture. For light chain cloning, the oligonucleotide 5'-ggt gtc gac GGA TAC AGT TGG TGC AGC ATC-3' (SEQ ID NO: 15), a primer corresponding to the kappa chain constant region, and the oligonucleotide 5'-MK 5'-cgg aag ctt GAY ATT GTG MTS ACM CAR WCT MCA-3' (SEQ ID NO: 16), a primer corresponding to the N-terminus of the kappa chain variable region, were used, respectively.For efficient cloning of polymerase chain reaction products, EcoRI restriction sites were added to the 5'-primer terminus and SalI restriction sites were added to the 3'-primer terminus for the heavy chain. For the light chain, HindIII restriction sites were added to the 5'-primer terminus and SalI restriction sites were added to the 3'-primer terminus. After mixing the polymerase reaction primers of the heavy or light chain and 1 unit of i-pfu DNA Polymerase (iNtRON Biotechnology, Seoul, Korea), the reaction was performed once at 95°C for 5 minutes, followed by 30 cycles of 95°C for 1 minute, 35°C for 1 minute, and 72°C for 1 minute. As a result, amplified DNA was obtained at a position corresponding to approximately 400 bp, which is estimated to be a DNA fragment corresponding to the heavy chain variable region, and approximately 390 bp, which is estimated to be a DNA fragment corresponding to the light chain variable region (Figures 13a and 13b).

[0199]

[0200] 8-2. Cloning and Sequence Analysis of the NPB15 Monoclonal Antibody Gene

[0201] In order to clone the NPB15 gene amplified in the above example, first, the polymerase chain reaction product was treated with EcoRI and SalI for the heavy chain and with HindIII and SalI for the light chain, and then developed on a 1.0% (w / v) agarose gel. DNA corresponding to approximately 400 bp and 390 bp was isolated using a FavorPrep GEL PCR Purification Kit (Favorgen, Pingtung, Taiwan). pBluescript KS+, which was to be used as a vector for cloning the heavy chain gene, was treated with EcoRI and SalI, and pBluescript KS+, which was to be used as a cloning vector for the light chain gene, was treated with HindIII and SalI, and then isolated using a FavorPrep GEL PCR Purification Kit. These two DNAs were ligated with T4 DNA ligase (New England Biolab, Massachusetts, USA) and transformed into E. coli DH5α using the CaCl2 method. For DNA sequence analysis of antibody genes, the above-mentioned multiple clones were cultured overnight in 3 ml of LB medium containing 100 μg / ml of ampicillin. Plasmid DNA was isolated using a DNA-Spin plasmid mini prep kit (Intron, Korea) according to the supplier's protocol. For the heavy chain, an E. coli clone with a DNA insert of approximately 400 bp in size was selected (Fig. 13c), and for the light chain, an E. coli clone with a DNA insert of approximately 390 bp in size was selected (Fig. 13d). The base sequence of each DNA insert was confirmed by nucleotide sequencing (Bionics, Seoul, Korea). After translating the base sequences of the heavy and light chain DNA into amino acids, the antigen recognition determining sites according to the antibody structure were organized using Kabat numbering. As a result, the heavy chain corresponded to subgroup II, and the light chain corresponded to subgroup V. The antigen-binding sites, CDR1, 2, and 3, are indicated in bold in each sequence (Figs. 14 and 15).

[0202]

[0203] Example 9. Preparation of expression vector for mouse-human chimeric Chi-NPB15 antibody

[0204] When mouse-derived monoclonal antibodies are used in humans, there is a possibility of causing immune side effects. To overcome this, mouse-derived monoclonal antibodies can be produced as mouse-human chimeric antibodies (Chimeric antibodies) with fewer immune side effects and used as therapeutic antibodies. To develop a mouse-human chimeric antibody (Chi-NB15) of the mouse-derived monoclonal antibody NPB15, the NPB15 antibody gene and the constant region of a human antibody were combined, and this was cloned into an expression vector to produce the Chi-NPB15 antibody expression vector for antibody production. First, the heavy and light chain genes of NPB15 obtained from the mouse were subjected to PCR (polymerase chain reaction) to isolate DNA corresponding to approximately 400 bp and 390 bp using a gel extraction kit (FAVORGEN, Taiwan). To synthesize a signal peptide sequence to be attached in front of the heavy chain gene, PCR was performed using the pdCMV-dhfr vector (Korean Patent No. 2120223) as a template and the primers h2B7-HC-SLIC-5`(5`-GCC AGT GTG CTG GAA TTC ACT CTA ACC-3`) (SEQ ID NO: 17) and NPB15-Chi-HC-SP-3`(5`-CTG CAC CTG GGA GTG GAC ACC TGT AGT TA-3`) (SEQ ID NO: 18). And, in order to amplify the heavy chain variable region of NPB15, PCR was performed using the DNA of the heavy chain variable region as a template, using the primers NPB15-Chi-HC-SP-5`(5`-GTC CAC TCC CAG GTG CAG CTG CAG-3`) (SEQ ID NO: 19) and NPB15-chi-HC-3`(5`-CCT TGG TGG AGG CTG AGG AGA CTG TGA G-3`) (SEQ ID NO: 20).Again, in order to link the heavy chain signal peptide sequence and the heavy chain variable region of the NPB15 antibody, the DNA prepared by the above PCR was mixed as a template and recombinant PCR was performed using the primers h2B7-HC-SLIC-5` and H2B7-HC-SLIC-3` (AAG ACC GAT GGG CCC TTG GTG GAG-3`) (SEQ ID NO: 21). The PCR product was developed on a 1% agarose gel, and approximately 450 bp of DNA linking the heavy chain variable region and the heavy chain signal peptide sequence was confirmed (Fig. 16a). This was isolated using a FavorPrep GEL™ PCR Purification Kit (Farvorgen, Taiwan), treated with EcoRI and ApaI, and then isolated again using a FavorPrep GEL™ PCR Purification Kit. This was inserted into the EcoRI and ApaI sites of the pdCMV-dhfr vector containing the human heavy chain constant region (IgG1) gene using T4 DNA ligase (NEB, USA) (Fig. 16b).

[0205] To synthesize a signal peptide sequence to be attached in front of the light chain gene, PCR was performed using the pdCMV-dhfr vector as a template, using the primer h2B7-LC-SLIC-5` (5`-ATA GGG AGA CCC AAG CTT CGG CAC GAG CAG A-3`) (SEQ ID NO: 22) and the primer NPB15-Chi-LC-SP-3` (5`-CAT CAC AAT ATC TCC TTC AAC ACC AGA CA-3`) (SEQ ID NO: 23) as a pair. And in order to amplify the light chain variable region of the NPB15 antibody, PCR was performed using the DNA of the light chain variable region as a template, the NPB15-Chi-LC-SP-5`(5`-GTT GAA GGA GAT ATT GTG ATG ACA CAG TC-3`) (SEQ ID NO: 24) primer, and Chi-NPB15-LC-SLIC-3`(5`-TGG TGC AGC CAC CGT ACG TTT GAT TTC CA-3`) (SEQ ID NO: 25) primer. Again, in order to link the light chain signal peptide sequence and the light chain variable region of the NPB15 antibody, the DNA prepared by the above PCR was mixed as a template, and recombinant PCR was performed using the h2B7-LC-SLIC-5` primer and the Chi-NPB15-LC-SLIC-3` primer. The PCR product was developed on a 1% agarose gel, and approximately 430 bp of DNA linking the light chain variable region and light chain signal peptide sequences was identified (Fig. 16c). This DNA was isolated using the FavorPrep GEL™ PCR Purification Kit, treated with HindIII and BsiwI, and then isolated again using the FavorPrep GEL™ PCR Purification Kit.This was cloned into the HindIII and BisWI sites of the pdCMV-dhfr-chi-NPB15-HC vector into which the heavy chain gene was inserted, and linked to the human light chain constant region (Ck) gene (Fig. 16d), and the final chimeric NPB15 antibody expression vector was named 'pdCMV-dhfr-chi-NPB15'. The final vector DNA was transformed into Escherichia coli DH5α using the RbCl2 method, and clones with heavy chain gene inserts of approximately 450 bp in size and E. coli clones with light chain gene inserts of approximately 430 bp in size were selected. For DNA base sequence analysis of the antibody genes, the above several clones were cultured overnight in 5 ml of LB medium containing 50 μg / ml of ampicillin, and then plasmid DNA was isolated using a DNA-spinTM Plasmid DNA purification kit (INTRON, Korea), and the base sequence of each DNA insert was confirmed (Bionics, Korea). The DNA base sequences of the heavy and light chain variable regions of pdCMV-dhfr-chi-NPB15 were identical to the gene sequence of the existing NPB15 variable region, and were confirmed to be correctly linked to the signal peptide sequence and human heavy and light chain constant region genes (Figs. 17 and 18).

[0206]

[0207] Example 10. Expression, purification, and activity analysis of mouse-human chimeric antibody Chi-NPB15.

[0208] The chimeric antibody linked to the NPB15 mouse variable region and human constant region was named Chi-NPB15, and to produce and purify this chimeric antibody, 1x10 human embryonic kidney cell line HEK293FT cell line was first cultured in a 150 mm culture dish. 7Cells were cultured in 20 ml of DMEM (Biowest, France) medium. 50 μg of the pdCMV-dhfr-Chi-NPB15 expression vector and 75 μl of polyethyleneimine (1 mg / ml) were mixed, then mixed with 500 μl of transfection optimization medium and spread onto the cell culture medium of the cultured cells. After culturing for 24 h, the cell culture medium was collected and the antibody was purified. The collected cell culture medium was added to a column filled with Protein G agarose beads (Amicogen, Korea) to allow the antibody to bind to the Protein G agarose beads. The beads were washed with PBS (pH 8.0), and the antibody was eluted from the Protein G agarose beads by adding 10 ml of 0.1 M glycine (pH 2.8) and 1 M Tris-HCl (pH 9.0). The purity of the purified Chi-NPB15 chimeric antibody was confirmed through SDS-PAGE and Coomassie blue staining (Fig. 19a). In addition, the chimeric antibody was detected through Western blotting using a goat anti-human antibody (goat-α-hIgG-HRP, Invitrogen, USA) as a secondary antibody, and it was confirmed that the chimeric antibody was an antibody containing the constant region of a human antibody, showing that a chimeric antibody having a mouse antibody variable region and a human constant region was properly produced (Fig. 19b). Next, in order to analyze whether the isolated Chi-NPB15 antibody exhibited antibody activity, the binding of NPB15 and chi-NPB15 antibody to the liver cancer cell line Huh7 was analyzed using a flow cytometer as in Example 3-1. As a result, it was observed that both antibodies equally well bound to Huh7 cells, confirming that the chimeric antibody Chi-NPB15 is an antibody with similar activity to NPB15 (Fig. 19c).

[0209]

[0210] Example 11. Antibody-dependent cytotoxicity of chimeric Chi-NPB15 antibody in hepatoma cells

[0211] The cell-dependent cytotoxicity (ADCC) of NPB15 was measured in the hepatoma cell line Huh7 using a chimeric Chi-NPB15 antibody, which has the variable region of the mouse antibody NPB15 and the human IgG1 constant region. For the measurement, 37,500 human hepatoma cell line Huh7 cells were dispensed into the inner 60 wells of a 96-well white plate (SPL) excluding the outer 36 wells the day before the experiment using 100 ㎕ of RPMI-1640 (Biowest) medium containing 10% FBS and cultured for 24 hours. The next morning, 95 ㎕ of the culture medium was removed, and 25 ㎕ of ADCC assay buffer (1.4 ml low IgG serum + 33.6 ml RPMI) included in the ADCC kit (Promega, G7010) was treated to the inner 60 wells and 75 ㎕ to the outer 36 wells, and serial dilutions of the antibody were performed. Serial dilutions were performed by diluting each hIgG isotype control and NPB15 human chimeric antibody by 1 / 3 from 100 μg / ml in ADCC assay buffer, and 10 wells were performed in triplicate for 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and no antibody. After treating each well with 25 μl of each antibody dilution, 25 μl of cell suspension prepared by diluting 630 μl ADCC bioassay effector cells thawing in 3.6 ml ADCC assay buffer was treated to set the E:T ratio to 12:1, and incubated for 6 hours at 37°C and 5% CO2. After 6 hours, the plate was left at room temperature for 15 minutes to lower the temperature, and 75 ㎕ of the previously prepared Bio-Glo luciferase assay reagent was treated to each of the inner 60 wells and 75 ㎕ to each of the outer 3 wells (blank control), and after 5 minutes of reaction, luminescence was measured.As a result of the experiment, compared to the negative control hIgG1 isotype antibody, it was confirmed that the luminescence of the Chi-NPB15 antibody significantly increased from a concentration of 5 μg / ml or higher (Fig. 20), suggesting that the NPB15 antibody can induce the death of liver cancer cells through ADCC via targeting CD98.

[0212]

[0213] Example 12. Analysis of the anticancer effect of NPB15 in a human liver cancer cell xenograft mouse model.

[0214] Based on the function of the NPB15 antibody confirmed in the previous examples, the possibility of using the NPB15 antibody for the prevention or treatment of CD98-expressing cancers was confirmed in vivo. To establish a liver cancer animal model, immunodeficient nude (Balb / c nu / nu) mice (Nara biotech, Korea) were purchased, and a liver cancer cell line xenograft nude mouse model was established to confirm cancer cell growth.

[0215] Huh7 cells, a liver cancer cell line, were detached with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea), neutralized with cell culture medium containing 10% FBS (VWR), centrifuged, and then cell counting was performed. 7.5X10 per mouse 6 A cell suspension was prepared by suspending the cells in 200 ㎕ of a 1:1 dilution of PBS (pH 7.4):Matrigel (Corning). The cell suspension was xenografted by subcutaneous injection into the dorsal flank of 12 5-week-old female Balb / C-nu nude mice (Nara Bio) that had been stabilized in a breeding room for 1 week.

[0216] 50 mm 3Among the mice in which tumors of the above size were observed, 6 mice were injected with mIgG1 isotype control antibody, and the other 6 mice were administered NPB15 at a dose of 10 mg / kg via tail vein injection at 2-day intervals for 20 days (Fig. 21a). The tumor volume on each date was calculated as 1 / 2*long axis*short axis. 2 It was measured by calculating.

[0217] As a result, we observed that mice administered NPB15 showed significantly suppressed tumor growth compared to mice administered mIgG1 as a control group (Fig. 21b). When comparing the sizes of tumors harvested from mice sacrificed on day 20 after antibody administration, we confirmed that the tumors harvested from mice administered NPB15 were smaller than those from the control group administered mIgG1 (Fig. 21c). In addition, when we compared the weights of tumor masses obtained from nude mice, we confirmed that the tumor weights of nude mice injected with NPB15 were smaller (Fig. 21d). Additionally, when we measured the body weights of mice to observe side effects due to antibody injection, there was no difference between the mIgG1 group and the NPB15 group (Fig. 21e). These results confirmed that NPB15 can reduce tumor growth in vivo without side effects and that NPB15 can be used for the prevention and treatment of cancer in vivo.

[0218]

[0219] Example 13. Analysis of the anticancer effect of Chi-NPB15 in a human hepatoma cell xenograft mouse model.

[0220] In addition to the functions of the NPB15 antibody confirmed in the previous examples, the Chi-NPB15 antibody, developed based on NPB15, was tested in vivo for its potential use in the prevention or treatment of CD98-expressing cancer cells. Human isotype control (Human IgG) and human-mouse chimeric antibody Chi-NPB15 were injected into a nude mouse model carrying a hepatoma cell line xenograft, and the inhibition of cancer cell growth was compared.

[0221] Huh7 liver cancer cells were detached using Tryple™ Express (Gibco, Seoul, Korea), neutralized with cell culture medium containing 10% FBS (VWR), centrifuged, and then cell counting was performed. 7.5X10 per mouse 6 A cell suspension was prepared by suspending the cells in 200 μl of a 1:1 dilution of PBS (pH 7.4):Matrigel (Corning). The cell suspension was injected subcutaneously into the dorsal flank of 5-week-old female Balb / C-nu nude mice (Nara Bio) that had been stabilized in a breeding room for 1 week, and xenografted. 50 mm 3 Among the mice in which the above cancers were observed, three mice were administered hIgG isotype control antibody and five mice were administered Chi-NPB15 chimeric antibody at a dose of 10 mg / kg each via tail vein injection for a total of seven times at 3-day intervals over 21 days (Fig. 22a). The tumor volume on each date was calculated as 1 / 2*long axis*short axis. 2 It was measured by calculating.

[0222] As a result, mice administered Chi-NPB15 also showed suppressed tumor growth compared to the control group administered hIgG (Fig. 22b). To observe any side effects caused by antibody injection, the body weights of the mice were measured, and no difference was observed between the hIgG and Chi-NPB15 groups (Fig. 22c). These results confirmed that Chi-NPB15 can reduce tumor growth in vivo without side effects, and that Chi-NPB15 can be used for the prevention and treatment of cancer in vivo.

Claims

1. An antibody comprising a heavy chain variable region comprising an HCDR1 consisting of an amino acid sequence of SEQ ID NO: 1, an HCDR2 consisting of an amino acid sequence of SEQ ID NO: 2, and an HCDR3 consisting of an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising an LCDR1 consisting of an amino acid sequence of SEQ ID NO: 4, an LCDR2 consisting of an amino acid sequence of SEQ ID NO: 5, and an LCDR3 consisting of an amino acid sequence of SEQ ID NO:

6.

2. An antibody according to claim 1, comprising a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 7 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO:

8.

3. In claim 1, the antibody is an antibody that binds to CD98 on the surface of pure human pluripotent stem cells, quasi-human pluripotent stem cells, human cancer cells, and cancer stem cells.

4. An antibody according to claim 1, further comprising a heavy chain constant region derived from a human IgG1 constant region and a light chain constant region derived from a human kappa constant region.

5. An antibody according to claim 1, comprising a heavy chain region consisting of an amino acid sequence of SEQ ID NO: 9 and a light chain region consisting of an amino acid sequence of SEQ ID NO:

10.

6. A composition for detecting undifferentiated pluripotent stem cells comprising an antibody according to any one of claims 1 to 5.

7. A pharmaceutical composition for preventing or treating cancer, comprising an antibody according to any one of claims 1 to 5.

8. A pharmaceutical composition for preventing or treating cancer, further comprising an anticancer substance conjugated to the antibody according to claim 7.

9. A pharmaceutical composition for preventing or treating cancer, wherein the anticancer substance according to claim 8 is conjugated via a secondary antibody that binds to the antibody.

10. A pharmaceutical composition for preventing or treating cancer, comprising at least one further selected from the group consisting of NK cells, macrophages, and neutrophils, according to claim 7.

11. A pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the cancer is a cancer derived from cancer cells that express CD98 protein on the cell membrane.

12. A pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the cancer is selected from the group consisting of liver cancer, lung cancer, embryonal carcinoma, pancreatic cancer, osteosarcoma, skin cancer, neuroblastoma, uterine cancer, prostate cancer, and colon cancer.

13. A hybridoma producing the antibody of claim 1.

14. A composition for diagnosing cancer comprising an antibody according to any one of claims 1 to 5.

15. A composition for diagnosing cancer according to claim 14, wherein the cancer is a cancer derived from cancer cells that express CD98 protein on the cell membrane.

16. A composition for diagnosing cancer according to claim 14, wherein the cancer is selected from the group consisting of liver cancer, lung cancer, embryonal carcinoma, pancreatic cancer, osteosarcoma, skin cancer, neuroblastoma, uterine cancer, prostate cancer, and colon cancer.

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