Novel Anti-mesothelin antibody and use thereof

Abstract

The present invention relates to: a novel anti-mesothelin antibody; and a pharmaceutical composition for treating cancer and a dual-targeting anticancer platform, comprising same. The mesothelin antibody according to an embodiment of the present invention has excellent mesothelin binding ability, and thus can be usefully used for cancer treatment.

Description

Novel anti-mesothelin antibodies and their uses The present invention relates to a novel anti-mesothelin antibody and a pharmaceutical composition for treating cancer comprising the same. Advances in medicine have led to the development of various anticancer drugs, broadly categorized as cytotoxic, targeted, and immunological. Cytotoxic drugs kill cancer cells through their toxicity, while immunological drugs enhance the immune system's ability to attack and destroy cancer cells. Targeted drugs target specific proteins in cancer cells. Unlike normal cells, cancer cells grow rapidly, but their growth can be amplified by signals from specific proteins. Targeted drugs detect and target these cancer cell signals, inhibiting their growth and proliferation. Mesothelin (MSLN) is a cell surface glycoprotein normally expressed in serous tissues such as the pleura, pericardium, and peritoneum. While not expressed in vital organs, it is frequently overexpressed in numerous solid tumors, including mesothelioma, pancreatic ductal adenocarcinoma, and ovarian cancer (Non-patent Document 1), making it an ideal target for cancer treatment. Additionally, mesothelin has recently attracted attention as a pancreatic cancer biomarker (Non-patent Document 2). [Non-patent literature] 1. Hassan, R., & Ho, M. (2008). Mesothelin targeted cancer immunotherapy. European journal of cancer (Oxford, England: 1990), 44(1), 46-53. 2. Lv, J., Li, P. Mesothelin as a biomarker for targeted therapy. Biomark Res 7, 18 (2019). Under the above circumstances, the present inventors have diligently sought to develop a novel antibody that specifically binds to human mesothelin. As a result, they have developed a novel antibody with high affinity for mesothelin, which is overexpressed in cancer cells, thereby completing the present invention. Furthermore, the present inventors have developed an anti-cancer platform comprising the anti-mesothelin antibody. Accordingly, one object of the present invention is to provide a novel anti-mesothelin antibody. Another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising the anti-mesothelin antibody or the anticancer platform. In order to achieve the above purpose, one aspect of the present invention is A heavy chain variable region comprising heavy chain CDR1 (complementarity determining region 1), CDR2, and CDR3, each represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and A mesothelin-specific antibody or antigen-binding fragment thereof is provided, comprising a light chain variable region comprising light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 57 and SEQ ID NO: 6, respectively. The present inventors performed ELISA screening on a scFv phage library using human mesothelin protein as an antigen to select clones that significantly bind to mesothelin (Figs. 1 and 2), and confirmed the sequence and complementarity determining regions (CDRs) of the scFv expressed in the clones, thereby completing the present invention. In the present invention, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically reacts with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, and antibodies in their entirety. The term also includes chimeric antibodies, humanized antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies. In the present invention, the term "antigen binding fragment" means a fragment having an antigen-antibody binding function within a complete antibody molecule, and may be selected from the group consisting of, for example, Fab (fragment antigen binding), Fab', F(ab')2, scFv (single-chain Fv), Fv (variable fragment), dsFv (disulfice-stabilized Fv fragments), diabody, Fd, Fd', and BiTE (bispecific T-cell engager), but is not limited thereto. Among the above antigen-binding fragments, Fab has a structure with a single antigen-binding site comprising variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the smallest antibody fragment that has only the heavy chain variable region (VH) and the light chain variable region (VL), and two-chain Fv has the heavy chain variable region and the light chain variable region linked non-covalently. Single-chain Fv (scFv) is a recombinant antibody fragment reconstructed by connecting only the variable region that constitutes the antigen-binding site from the light and heavy chains with a linker. Antibodies are large molecules of approximately 150 kDa, and they have difficulty working effectively in large tumors or tissues that are difficult to penetrate. However, scFv is a molecule of approximately 30 kDa, and is the smallest antibody fragment that maintains a complete antigen-binding site, so it maintains the antibody's unique specific antigen-binding ability and can work effectively in large tumors or tissues that are difficult to penetrate. Fd is an antibody fragment consisting of the variable region (VH) and the first constant region (CH1) domains of the heavy chain, and a bispecific T-cell engager (BiTE) is an antibody designed to simultaneously bind to a specific antigen and a T-cell surface protein, for example, it may be composed of two scFv molecules that recognize a surface antigen on a cancer cell and CD3 on a T cell, respectively. The above antigen-binding fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of a whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology. In the present invention, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region in the heavy and light chains of an antibody. The heavy and light chains may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for binding of the antibody to an antigen or epitope. According to one specific example of the present invention, the human mesothelin-specific antigen-binding fragment may be a scFv and may include a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 4 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 7. In addition, the human mesothelin-specific scFv may have a light chain variable region and a heavy chain variable region connected by a peptide linker, and a sequence of SEQ ID NO: 11 (GGGSSRSSSSGGGGSGGGG) may be used as the peptide linker. According to one specific example of the present invention, the human mesothelin-specific scFv may comprise a six histidine tag (6x His-tag) and an HA tag at the C-terminus for purification and tracking (Figure 3). In addition, the human mesothelin-specific scFv can bind to human mesothelin with a dissociation constant (Kd) of 11.4 nM. The dissociation constant of an antibody indicates its binding affinity to an antigen, and the lower the dissociation constant, the higher the binding affinity between the antibody and the antigen. The binding affinity can be determined by a conventional method. Another aspect of the present invention provides a polynucleotide encoding the human mesothelin-specific antibody or an antigen-binding fragment thereof, an expression vector comprising the polynucleotide, and a transformant into which the expression vector has been introduced. According to one specific example of the present invention, the polynucleotide coating the human mesothelin-specific scFv may include a nucleic acid sequence represented by SEQ ID NO: 8, and the polynucleotide encoding the human mesothelin-specific antibody may include, but is not limited to, nucleic acid sequences represented by SEQ ID NOs: 9 and 10. Meanwhile, a vector comprising a polynucleotide encoding a human mesothelin-specific antibody may comprise a polynucleotide encoding each of the heavy chain and light chain of the antibody, or a polynucleotide encoding both the heavy chain and the light chain. When the polynucleotides encoding the heavy chain and light chain of the antibody are present in separate vectors, the heavy chain encoding vector and the light chain encoding vector may be introduced into a single transformant or into separate transformants. When the vectors are introduced into separate transformants, the expressed antibody light chain and antibody heavy chain can be recovered and produced as an intact antibody. In the present invention, the expression vector comprising a polynucleotide encoding the human mesothelin-specific antibody or an antigen-binding fragment thereof may be a vector capable of replicating and / or expressing the polynucleotide in prokaryotic or eukaryotic cells, including, but not limited to, bacterial cells (e.g., Escherichia coli, etc.), mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, or insect cells. Preferably, the expression vector may be a vector that is operably linked to an appropriate promoter so that the polynucleotide can be expressed in a host cell and includes at least one selectable marker. For example, the polynucleotide may be introduced into a phagemid, plasmid, cosmid, mini-chromosome, virus, or retrovirus vector. In the present invention, the transformant into which the expression vector has been introduced is not particularly limited, but may be a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella Typhimurium transformed by introducing the expression vector; a yeast cell; a fungal cell such as Pichia pastoris; an insect cell such as Drosophila or Spodoptera Sf9 cells; an animal cell such as CHO (Chinese hamster ovary cells), HEK (human embryonic kidney cells), or PERC.6 (human retinal cells); or a plant cell. The polynucleotide or the expression vector containing the same can be delivered into a host cell / transformant using a delivery method widely known in the art. For example, if the host cell is a prokaryotic cell, the CaCl2 method or electroporation can be used, and if the host cell is a eukaryotic cell, the microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, and gene bombardment method can be used, but are not limited thereto. Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the mesothelin-specific antibody or an antigen-binding fragment thereof as an active ingredient. While mesothelin is expressed at low levels in normal tissues, its overexpression has been confirmed in various solid tumors, including mesothelial cancer, pancreatic cancer, and ovarian cancer, and it is being studied as an anticancer target. For example, Korean Patent No. 10-2070016 discloses a chimeric antigen receptor containing a binding domain that specifically binds mesothelin and the anticancer effects of T cells expressing it. Therefore, mesothelin-specific antibodies could be useful for anticancer applications. The cancer may be selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, peritoneal cancer, endometrial cancer, stomach cancer, colon cancer, breast cancer, kidney cancer, thyroid cancer, prostate cancer, bile duct cancer, cervical cancer, esophageal cancer, thymic cancer, and blood cancer. In addition to containing a mesothelin-specific antibody or an antigen-binding fragment thereof as an active ingredient, the above pharmaceutical composition may further contain appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceuticals. The pharmaceutical composition according to the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerol gelatin. The composition of the present invention can be administered orally or parenterally, and any parenteral administration method can be used. Systemic or local administration is possible, but systemic administration is more preferred, and intravenous administration is most preferred. The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration of the administration, but can be appropriately selected by those skilled in the art. Administration may be administered once daily or in several divided doses. The above dosage does not limit the scope of the present invention in any way. Another aspect of the present invention provides a tumor-targeting antibody fusion protein comprising an antibody that specifically binds to mesothelin; an MMP cleavage site; an anticancer peptide and a cell-penetrating peptide sequentially linked thereto, wherein the MMP cleavage site comprises an amino acid sequence that is cleaved by MMP (Matrix metalloproteinase). According to one specific example of the present invention, the antibody specifically binding to mesothelin may be selected from the group consisting of the following, but in addition, all antibodies known as mesothelin-specific antibodies in the technical field to which the present invention pertains may be used. a) An antibody (1T1G5) comprising a heavy chain represented by the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34; b) an antibody (CSA0682) comprising a heavy chain represented by the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36; and c) An antibody (Anetumab) comprising a heavy chain represented by the amino acid sequence of SEQ ID NO: 37 and a light chain comprising the amino acid sequence of SEQ ID NO: 38. According to one specific example of the present invention, the MMP cleavage sequence may be a sequence represented by the amino acid sequence of SEQ ID NO: 25 or 26, and the anticancer peptide may be a sequence consisting of the amino acid sequence of SEQ ID NO: 14 or 15. According to one specific embodiment of the present invention, the tumor-targeting antibody fusion protein primarily targets tumors with a mesothelin antibody and induces tumor cell death with an anticancer peptide. To enable the anticancer peptide to enter cells, the anticancer peptide may be bound to a cell-penetrating peptide comprising any one amino acid sequence selected from SEQ ID NOs: 16 to 20 at the C-terminus. Another aspect of the present invention provides an expression vector comprising a polynucleotide encoding a tumor-targeting antibody fusion protein. According to one specific example of the present invention, the expression vector can express a second fusion protein comprising MBP (maltose-binding protein), TEV protease (Tobacco etch virus protease), a TEV protease cleavage sequence (cleavage site), and the tumor-targeting antibody fusion protein. Specifically, the second fusion protein may be a fusion protein in which MBP, TEV protease, a TEV protease cleavage sequence, and a tumor-targeting antibody are sequentially linked. According to one specific example of the present invention, the MBP may comprise an amino acid sequence represented by SEQ ID NO: 21, and the TEV protease may comprise an amino acid sequence represented by SEQ ID NO: 22. In addition, the TEV protease cleavage sequence may comprise an amino acid sequence represented by SEQ ID NO: 23. In general, scFv (or tumor-targeting antibody fusion protein containing scFv) is mostly expressed insoluble when expressed in E. coli, and there is a problem that the amount of protein expressed that can be functionally used is very small. Therefore, in order to improve the production yield of scFv (or tumor-targeting antibody fusion protein containing scFv) in E. coli, fusion of pelB sequences and the like have been attempted, but the effect was not great. In addition, although fusion of MBP was efficient in solubilizing scFv (or tumor-targeting antibody fusion protein containing scFv), an additional purification process was required to separate pure scFv (or tumor-targeting antibody fusion protein containing scFv) from the fusion protein, which was cumbersome and time-consuming. Accordingly, the present inventors have designed an expression system capable of purifying a soluble scFv (or a tumor-targeting antibody fusion protein containing the scFv) from E. coli in one step without any additional process. According to the expression vector of the present invention and the protein production method using the expression vector, the target protein can be produced homogeneously and soluble in E. coli, and since the target protein is cleaved by TEV protease expressed within the cell, there is an effect of purifying only the pure target protein in one step. Another aspect of the present invention provides a tumor-targeting antibody fusion protein comprising a whole antibody; an MMP cleavage site; an anti-cancer peptide and a cell-penetrating peptide sequentially linked thereto, wherein the MMP cleavage site comprises an amino acid sequence that is cleaved by MMP (Matrix metalloproteinase). In the present invention, the whole antibody refers to an antibody composed of a light chain and a heavy chain including both a variable region and a constant region. According to one specific example of the present invention, the whole antibody includes, but is not limited to, a mesothelin antibody, a HER2 targeting antibody, and an EGFR targeting antibody. According to one specific example of the present invention, the HER2 targeting antibody may be trastuzumab comprising a heavy chain represented by SEQ ID NO: 49 and a light chain represented by SEQ ID NO: 50. Additionally, the EGFR targeting antibody may be cetuximab comprising a heavy chain represented by SEQ ID NO: 53 and a light chain represented by SEQ ID NO: 54. Another aspect of the present invention provides a host cell transformed with an expression vector for the tumor-targeting antibody fusion protein. Methods for introducing exogenous nucleic acids into a host cell are well known in the art and will vary depending on the host cell used. Another aspect of the present invention provides a pharmaceutical composition for treating cancer comprising the tumor-targeting antibody fusion protein, and all of the contents described with respect to the pharmaceutical composition comprising the mesothelin antibody can be applied or applied as is to the pharmaceutical composition for treating cancer comprising the tumor-targeting antibody fusion protein. An antibody according to an example of the present invention has excellent binding ability to human mesothelin and can therefore be used for the treatment of various cancers, including pancreatic cancer, and in a dual-target anticancer platform. Figure 1 shows the results of identifying phage clones that bind to human mesothelin using a scFv phage library. Figure 2 shows the results of ELISA screening to identify phage clones that bind to human mesothelin. Figure 3 is a schematic diagram of a vector for expressing a single chain variable fragment (scFv) that specifically binds to human mesothelin. Figure 4 is a schematic diagram of a vector for expressing the light and heavy chains of a monoclonal antibody (designated mAb-1T1G5) that specifically binds to human mesothelin. Figure 5 shows the results of identifying scFv (designated 1T1G5; A) and monoclonal antibody (mAb-1T1G5; B and C) that specifically bind to human mesothelin expressed and purified in E. coli. Figure 6 shows the results of ELISA confirming the mesothelin-specific binding ability of 1T1G5. Figure 7 shows the results of confirming the fluorescence signal of the antibody after reacting mesothelin-positive cell lines with different concentrations of 1T1G5. Figure 8 shows the results of confirming the fluorescence signal of the antibody after reacting mesothelin-positive cell lines with different concentrations of mAb-1T1G5. Figure 9 is a summary diagram briefly illustrating the tumor-targeting antibody fusion protein platform of the present invention and the method for producing the same. Figure 10 shows the results of an experiment on the cell killing ability of a fusion protein in which various peptides (iRGD, dNP2, mdNP2, mmdNP2, or 9R) expected to have cell penetration ability are fused to the anticancer peptide (ACP or KLAK) of the present invention. Figure 10a shows the results of an experiment on the cell killing ability of the fusion protein against cancer cells, and Figure 10b is a graph showing the cell killing ability of the fusion protein against cancer cells in numerical form. Figure 11 shows the results of additional experiments on the cell killing ability of fusion proteins that fuse various peptides (TAT, Amtd, Pep1, dNP2, 9R, and iRGD) expected to have cell penetrating ability to an anticancer peptide (ACP or KLAK). Figure 12 is a schematic diagram showing a tumor-targeting antibody fusion protein that omits the MMP cleavage sequence and fuses a tumor-targeting delivery system, an anti-cancer peptide, and a cell-penetrating peptide. Figure 13 shows the results of confirming the in vitro anticancer activity of a fusion protein created by directly fusing cpACP to an antibody fragment or by fusing it via a cathapsin B cleavage site. Figure 14 shows the results of confirming the in vitro anticancer activity of the fusion protein by overlapping cpACP, which exhibits anticancer activity. Figure 15 shows the results of confirming the expression of a tumor-targeting antibody fusion protein that omits the MMP cleavage sequence and fuses a tumor-targeting delivery system, an anticancer peptide, and a cell-penetrating peptide, using SDS-PAGE. Figure 16 is a graph showing the binding affinity of a tumor-targeting antibody fusion protein that omits the MMP cleavage sequence and fuses a tumor-targeting delivery system, an anticancer peptide, and a cell-penetrating peptide to the target antigen. Figure 17 shows the results showing the apoptotic effect on cancer cells of a tumor-targeting antibody fusion protein that omits the MMP cleavage sequence and fuses a tumor-targeting delivery system, an anticancer peptide, and a cell-penetrating peptide. Figure 18 is a diagram showing tumor-targeting antibody fusion proteins (D303 and D305) that fuse a tumor-targeting delivery system, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide, confirmed by SDS-PAGE. Figure 19 shows the results of confirming the cell death effect of fusion proteins (D303 and D305) on cancer cells. Figure 20 is a diagram showing the fusion proteins (D301, D302, D303, and D304) confirmed by SDS-PAGE. Figure 21 shows the results of confirming the cell death effect of fusion proteins (D301, D302, D303, and D304) on cancer cells. Figure 22 shows the results of SDS-PAGE analysis of the expression of tumor-targeting antibody fusion proteins (D310, D311, D312, D313, D314, D315, D316) that fused a tumor-targeting delivery system, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide: M-marker, T-total cell lysate (E. coli cell lysate); P-pellet (precipitate obtained by centrifuging T); S-supernatant (supernatant obtained by centrifuging T); BP-Bind Pass (proteins not bound to resin); and E-elution (proteins eluted from resin). Figure 23 shows the results of SDS-PAGE analysis of the expression of tumor-targeting antibody fusion proteins (D310, D311, D312, and D314) that fuse a tumor-targeting delivery system, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide. Figure 24 is a diagram showing the results of specificity and affinity analysis of fusion proteins (D310, D311, D312, and D314) for HER2 antigen or HSA (serum albumin) protein. Figure 25 shows the results showing the apoptotic effect of fusion proteins (D310, D311, D312, and D314) on cancer cells. Figure 26 shows the results of confirming the anticancer activity of D311 in various tumor cell lines, immune cell lines, and general cell lines. Figure 27 shows the results of confirming tumor growth after administering fusion protein (D310 or D311) to SKOV3 transplanted nude mice. Figure 28 is a diagram showing a tumor growth curve over time in a SKOV3 transplant mouse model after administration of a fusion protein (D310, D311) according to one embodiment of the present invention. Figure 29 is a schematic diagram showing how the DTAT-MSLN platform using anti-mesothelin antibodies works. Figure 30 shows the expression vector (A), expression results (B), and mass spectrometry results (C) of three anti-mesothelin antibodies: 1T1G5, CSA0682 (0682), and anetumab (Ane). Figure 31 shows the results of ELISA and flow cytometry confirming the affinity of three anti-mesothelin antibodies, 1T1G5, CSA0682 (0682), and anetumab (Ane), for mesothelin. Figure 32 shows the DTAT-MSLN platform expression vector and DAR (drug antibody ration) using anti-mesothelin antibodies. Figure 33 shows the results of confirming the expression of three types of DTAT-MSLN (DTAT-1T1G5, DTAT-0682, and DTAT-Ane). Figure 34 shows the results of confirming the expression levels of three types of DTAT-MSLN (DTAT-1T1G5, DTAT-0682, and DTAT-Ane). Figure 35 shows the results of confirming the affinity of three types of DTAT-MSLN (DTAT-1T1G5, DTAT-0682, and DTAT-Ane) for mesothelin (MSLN) and megakaryocyte stimulating factor (MPF). Figure 36 shows the results comparing the affinity of three DTAT-MSLNs (DTAT-1T1G5, DTAT-0682, and DTAT-Ane) to mesothelin (MSLN) with anti-mesothelin antibodies. Figure 37 shows the results of confirming the affinity of three types of DTAT-MSLN (DTAT-1T1G5, DTAT-0682, and DTAT-Ane) for mesothelin (MSLN) using ELISA and FACS. Figure 38 shows the results of confirming the degree of cell death by IC50 after treating cancer cell lines (Aspc-1, BxPC-3, Capan1, Capan2, MIA-Paca2, Panc1, SNU-231, SKOV3, and A431), immune cell line (THP-1), and general cell line (CHOs) with DTAT2-1T1G5. Figure 39 shows the results of confirming the degree of cell death by IC50 after treating cancer cell lines (Aspc-1, BxPC-3, Capan1, Capan2, MIA-Paca2, Panc1, SNU-231, SKOV3, and A431), immune cell line (THP-1), and normal cell line (CHOs) with DTAT2-0682. Figure 40 shows the results of confirming the degree of cell death by IC50 after treating cancer cell lines (Aspc-1, BxPC-3, Capan1, Capan2, MIA-Paca2, Panc1, SNU-231, SKOV3, and A431), immune cell line (THP-1), and normal cell line (CHOs) with DTAT2-Ane. Figure 41 shows the results of checking the degree of degradation after storing three types of DTAT-MSLN (DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane) in buffer (0.1 M Tris-glycine, pH 7.6). Figure 42 shows the results of examining the degree of degradation after storing three types of DTAT-MSLN (DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane) in human serum. Figure 43 shows the results of ELISA to determine the degree of degradation of three types of DTAT-MSLN (DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane) stored in buffer (0.1 M Tris-glycine, pH 7.6). Figure 44 shows the results of ELISA to determine the degree of degradation after storing three types of DTAT-MSLN (DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane) in human serum. Figure 45 shows the results of confirming the effects after administering three types of DTAT-MSLN (DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane) to tumor-induced BALB / c nude mice: A - administration schedule; B - tumor size measurement results; C - tumor volume measurement results; and D - body weight measurement results. Figure 46 shows the results of confirming whether DTAT4-0682 is expressed in its entirety in HEK293T cells in which the CS (Cancer-Specific cleavage seq.) gene is knocked out. Figure 47 shows the results comparing the cell death efficiency of DTAT2-0682 produced in another cell line (CHO-S / 293T). Figure 48 shows the results of confirming the expression of DTAT4-0682 while changing the purified resin and cell stabilizer in CHO-S. Figure 49 shows the results of analyzing the affinity of six DTAT2 / 4-MSLNs for each monoclonal antibody: A-ELISA; and B-FACS analysis. Figure 50 shows the results of comparing the cell death efficiency by IC50 and IC80 after treating AsPC1 cells with DTAT2-0682 and DTAT4-0682. Figure 51 shows the production process of DTAT-Tra using trastuzumab: A - expression vector, B - expression confirmation results; C - HEK293T cells with CS gene knockout; D - DTAT-Tra produced in HEK293T cells with C-CS gene knockout; E - results of expressing DTAT-Tra in increasing glycerol concentrations; and F - results of expressing DTAT-Tra under 20% glycerol conditions. Figure 52 shows the results of confirming the characteristics of DTAT-Tra: A-mass spectrometry results; B-affinity confirmation results for HER2; C-SKOV3 cell affinity confirmation results. Figure 53 shows the results confirming the anticancer activity of DTAT2-Tra and DTAT4-Tra. Figure 54 shows the results of confirming anticancer activity after treating cancer cell lines, immune cell lines, and general cell lines with DTAT2-Tra and DTAT4-Tra. Figure 55 shows the results of inducing mutations in each motif constituting DTAT2-Tra (A), the results of confirming the expression of the mutant (B), and the results of confirming the anticancer activity of the mutant (C). Figure 56 is a schematic diagram showing the principle by which DTAT-Tra exhibits anticancer activity. Figure 57 is a schematic diagram schematically showing the composition and operating principle of DTAT-Cet. Figure 58 shows the production process of DTAT-Cet using cetuximab: A - expression vector, B - expression confirmation result; C - DTAT-Cet expression result while increasing glycerol concentration; Figure 59 shows the production results and comparison of anticancer effects of DTAT-Cet: A - Results of expression with the addition of 20% glycerol; B - Results of confirming production efficiency when expression was with the addition of 20% glycerol; C - Results of comparison of anticancer activities of DTAT2-Cet and DTAT4-Cet. Figure 60 shows the results of confirming the characteristics of DTAT-Cet: A-mass spectrometry results; B-affinity confirmation results for EGFR1; and Affinity confirmation results for A431 cells. Figure 61 shows the results of confirming anticancer activity after treating cancer cell lines, immune cell lines, and general cell lines with DTAT2-Cet and DTAT4-Cet. Figure 62 shows the results of inducing mutations in each motif constituting DTAT2-Cet (A), the results of confirming the expression of the mutant (B), and the results of confirming the anticancer activity of the mutant (C). Figure 63 is a schematic diagram showing the principle by which DTAT-Cet exhibits anticancer activity. Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples. Example 1: Screening for human anti-mesothelin antibodies 1-1. Phage display We constructed an M13 bacteriophage library expressing the variable regions (heavy and light chain variable regions) of various antibodies on its surface as single-chain variable fragments (scFv). Antibodies were then selected through a panning process. Panning is the process of selecting antibodies that bind to a specific antigen from a diverse antibody pool. Five rounds of panning were used to select antibody candidates that selectively bind to mesothelin. The detailed process is as follows. 1) Mesothelin antigen was coated on a tube (immune tube) at a concentration of 1 μg / ml (coating solution: sodium bicarbonate buffer (pH9.6)). 2) The tube was washed with PBS (PBST) containing 0.05% (v / v) Tween 20 to remove mesothelin not bound to the tube. 3) Block the tube with 3% skim milk. 4) The phage library was added to the tube (1st and 5th: 1 x 10^13 <, 2nd to 4th: 1 x 10^12 <) to induce binding of phage to mesothelin. 5) The tube was washed to remove phages that did not bind to mesothelin. 6) A phage showing affinity for mesothelin was isolated. 7) The isolated phage was amplified by infecting host cells or bacteria. 8) Returning to step 1), steps 2) and 3) were repeated to identify the optimal binding sequence. Subsequently, the phage repertoire showing affinity for mesothelin was concentrated and purified using a precipitation method, and the phage titer was increased. The results of the phage display are shown in Fig. 1. 1-2. ELISA screening ELISA screening was performed to isolate antibodies that specifically bind to mesothelin on an independent clonal basis. The phages selected in Example 1-1 were added to plates coated or uncoated with mesothelin antigen, and the absorbance was measured at OD450. Phages for which the difference in absorbance measured on the mesothelin-coated plate was 1 or more were selected. Screening results identified two phages that specifically bind to mesothelin (Fig. 2). Furthermore, sequencing results confirmed that the scFvs expressed by the two phages were a single clone. In Figure 2, D31 is the absorbance measured on the antigen-coated plate, Background control (BC) is the absorbance measured on the plate without the antigen coating, and delta is the difference in absorbance between the two plates. Example 2: Production of antibody expression vector 2-1. Production of anti-mesothelin antibody expression vector The antibody sequences confirmed in Example 1-2 are listed in Table 1. SEQ ID NO: DESCRIPTION Amino acid sequence 1HCDR1GFTFSSYA2HCDR2ISGSGGST3HCDR3AKRVITLDY4variable heavy (VH) chain fragmentQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRVITLDYWGQGTLVTVSS5LCDR1SSNIGSNY57LCDR2RNNQ6LCDR3GTWDSSLSAVV7variable light (VL) chain fragmentLTQPPSVSGSPGQSITISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDSSLSAVVFGGGTQLTVL The sequences in Table 1 above were introduced into the vectors disclosed in Figures 3 and 4 to construct scFv and monoclonal antibody (mAb) expression vectors that specifically bind to mesothelin. The monoclonal antibody expressed the light chain and heavy chain sequences in separate vectors. 2-2. Antibody expression and purification - scFv production and purification The vector constructed in Example 2-1 was transformed into Escherichia coli BL21(DE3) using the heat shock method. The transformed BL21(DE3) was spread on LB agar medium containing ampicillin and cultured at 37°C for more than 16 hours. One colony of BL21 (DE3) was inoculated onto LB medium containing ampicillin and cultured until OD 600 nm reached 1.0. Protein expression was then induced by treatment with 1 mM IPTG, and the culture was further cultured for 16 hours. After the culture was completed, the culture medium was centrifuged to recover only the cells, which were then lysed by sonication. The cell lysate was centrifuged to recover only the supernatant. The supernatant was subjected to affinity chromatography using Ni-NTA agar resin to purify the scFv (hereinafter referred to as 1T1G5). - Production and purification of mAb-1T1G5 The vector shown in Figure 4 was introduced into CHO-S cells via a lipid-based transfection method. Protein expression was induced by culturing the cells for 5 days at 37°C, 8% CO2, and ≥80% humidity. After 5 days, the culture medium was centrifuged, and the supernatant was recovered. The supernatant was passed through Protein A resin for affinity chromatography, and the anti-mesothelin monoclonal antibody (hereinafter referred to as mAb-1T1G5) was purified. - Antibody purification results The sizes of purified 1T1G5 and mAb-1T1G5 were determined. The predicted sizes of 1T1G5 and mAb-1T1G5 are 26.3 kDa and 143.5 kDa, respectively. In addition, the predicted sizes of mAb-1T1G5 HC (heavy chain) and mAb-1T1G5 LC (light chain) are 48.9 kDa and 22.8 kDa, respectively. As a result of the verification, it was confirmed that both 1T1G5 and mAb-1T1G5 had the expected size (Fig. 5). Example 3: Confirmation of the antibody's ability to bind to mesothelin 3-1. ELISA (Enzyme-Linked Immunosorbent Assay) Mesothelin as an antigen and HSA (human serum albumin) as a negative control were added to each well at 50 ng per well in 24 wells and coated at 4℃ for 16 hours. Bicarbonate / carbonate coating buffer (100 mM, pH 9.6) was used as the coating buffer. Afterwards, the coating buffer and antigen solution were removed, and each well was washed three times with 100 ㎕ of PBST (PBS + 0.05% (v / v) Tween 20). After treating each well with 200 ㎕ of blocking buffer (PBS + 1% (w / v) BSA), the plate was stored at room temperature for 1 hour. After removing the blocking buffer, 1T1G5, serially diluted ½ each in 24 steps from 500 nM to 0 nM with PBS, was treated to each well and the plate was stored at room temperature for 1 hour. The PBS+1T1G5 solution was removed, and each well was washed four times with 100 μl of (PBS+0.05% (v / v) Tween 20). The secondary antibody (anti-HA-HRP conjugated) was diluted 4000:1 in PBS, and 100 μl was added to each well, and the plate was stored at room temperature for 1 hour. The PBS+secondary antibody solution was removed, and each well was washed four times with 100 μl of PBST (PBS+0.05% (v / v) Tween 20). 100 μl of TMB was added to each well, and the plate was stored at room temperature for 5 minutes. The reaction was stopped by adding 100 μl of 1 M HCl to each well. The absorbance was measured at 450 nm. The measurement results showed that 1T1G5 is a mesothelin-specific antibody that binds only to mesothelin and not to HSA. In particular, 1T1G5 demonstrated excellent binding affinity, with a calculated Kd value of 11.4 nM (Fig. 6). 3-2. Flow cytometry - Experimental group 1. 1T1G5 combined with MSLN+ cell line MIA PaCa-2 / hMSLN 2. mAb-1T1G5 combined with MSLN+ cell line MIA PaCa-2 / hMSLN -Experimental method MIA PaCa-2 / hMSLN cells, a mesothelin-positive cell line, were prepared in FACS buffer (PBS+2% FBS). 1T1G5 and mAb-1T1G5 were serially diluted and treated with 10^6 cells at each concentration for 1 hour at 4°C. Cells were harvested by centrifugation at 500g for 3 minutes at 4°C, and then washed three times with 100 μL of PBS. The secondary antibody of 1T1G5, Anti-HA-tag-Dylight 650, was diluted 1:3000 in PBS, and the secondary antibody of mAb-1T1G5, ProteinA-FITC, was diluted 1:1000 in PBS. 100 μl of the diluted secondary antibodies were added to each cell and stored at 4°C for 1 h. The cells were harvested by centrifugation at 500 g for 3 minutes at 4°C, and washed four times with 100 μl of PBS. The cells were resuspended in 300 μl of FACS buffer and analyzed using a flow cytometry instrument (1T1G5: 650 / 660 nm; mAb-1T1G5: 495 / 519 nm). Our analysis results confirmed that both 1T1G5 and mAb-1T1G5 bound to MSLN-positive cell lines (Figs. 7 and 8). Example 4: Fusion of anticancer peptides and cell-penetrating peptides An anticancer peptide with tumor cell killing ability was fused with a cell penetrating peptide so that it can penetrate tumor cells and target intracellular target proteins. For the above purpose, cell-penetrating anticancer peptides A to J in which the anticancer active peptide sequence ACP (SEQ ID NO: 14) or KLAK (SEQ ID NO: 15) and the cell-penetrating peptide sequence iRGD (SEQ ID NO: 16), dNP2 (SEQ ID NO: 17), mdNP2 (SEQ ID NO: 18), mmdNP2 (SEQ ID NO: 19), or 9R (SEQ ID NO: 20) are linked, and an acetyl group and an amide group are attached to the N-terminus and C-terminus, respectively, and the entire amino acid sequence was synthesized by requesting Peptmic Co., Ltd. Ovarian cancer cell line SKOV-3 was seeded in 96-well plates with 90 μl of culture medium at 4,000 cells / well each, and then cultured at 37°C and 5% CO2 for 4 hours. 10 μl of synthetic peptides A to J were added to each well plate, and treated once at concentrations of 0, 0.15, 0.31, 0.63, 1.3, 2.5, 5, and 10 μM, respectively, and incubated for 36 h at 37°C and 5% CO2. After incubation, 10 μl of CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) solution was treated to the well plate, and the reaction was performed for 2 h at 37°C and 5% CO2. After the reaction was completed, the absorbance was measured using a microplate reader. Specific combinations of anticancer peptides and cell-penetrating peptides are shown in Table 2 below. Classification Anticancer peptide Cell penetrating peptide AACP (SEQ ID NO: 14) iRGD (SEQ ID NO: 16) BdNP2 (SEQ ID NO: 17) CmdNP2 (SEQ ID NO: 18) DmmdNP2 (SEQ ID NO: 19) E9R (SEQ ID NO: 20) FKLAK (SEQ ID NO: 15) iRGD (SEQ ID NO: 16) GdNP2 (SEQ ID NO: 17) HmdNP2 (SEQ ID NO: 18) ImmdNP2 (SEQ ID NO: 19) J9R (SEQ ID NO: 20) Figure 10a shows the results of an experiment to determine the cell killing ability of the fusion protein against cancer cells, and Figure 10b is a graph that quantifies the cell killing ability of the fusion protein against cancer cells. Looking at the above Figure 10, it can be confirmed that the anticancer peptide ACP has an excellent cell killing ability against cancer cells when fused with iRGD ("A"), and the anticancer peptide KLAK has an excellent cell killing ability against cancer cells when fused with dNP2, mdNP2, mmdNP2, and 9R ("G", "H", "I", "J"). The superior anticancer efficacy of the ACP-iRGD combination was also confirmed in other experiments. SK-OV-3 cells were treated with various concentrations of the anticancer peptides ACP or KLAK in combination with various cell-penetrating peptides, and cell viability was assessed. The results showed that the ACP-iRGD combination exhibited significantly superior efficacy (Figure 11). Example 5: Fusion of a tumor-targeting delivery system, an anticancer peptide, and a cell-penetrating peptide (scFv-ACP-iRGD) By sequentially linking an anticancer peptide and a cell-penetrating peptide to the tumor-targeting delivery system of the present invention, a fusion protein was manufactured, and then the binding affinity of the fusion protein to the target antigen and the cell killing ability against cancer cells were confirmed. 5-1. Preparation of a fusion protein combining a tumor-targeting delivery system, an anticancer peptide, and a cell-penetrating peptide. A fusion protein was prepared by sequentially linking an anticancer peptide and a cell-penetrating peptide to a tumor-targeting delivery system (Figure 12). The specific sequences of the components constituting each fusion protein are shown in Table 3 below. Classification Amino acid sequence MBP Sequence number 21 TEV protease Sequence number 22 TEV protease cleavage sequence Sequence number 23 scFv Sequence number 24 Myc His-Tag (8xH) “HHHHHHHH” ACP Sequence number 14 iRGD Sequence number 16 In the above Table 3, “scFv” is a recombinant antibody (SEQ ID NO: 24) in which the variable region of the light chain (SEQ ID NO: 39) and the variable region of the heavy chain (SEQ ID NO: 40) of an anti-HER2 antibody (Trastuzumab) targeting HER2 are linked by a linker sequence (SEQ ID NO: 41). Specifically, the amino acid sequence corresponding to the structure of the [TEV cleavage sequence-scFv-ACP-iRGD] of the above fusion protein was specified, and the nucleic acid sequence encoding the amino acid sequence was optimized with codons suitable for expression in E. coli to determine the nucleic acid sequence. In addition, for future deletion of ACP and iRGD, adjacent XbaI site and SalI site were added to each sequence, and a stop codon was added to the terminal. Afterwards, for cloning, SacI site and PstI site were added to the terminal, and the entire nucleic acid sequence was synthesized by requesting Cosmogenetech Co., Ltd. Meanwhile, when cpACP was fused directly to an antibody fragment or fused via the cathapsin B cleavage site, no in vitro anticancer activity was observed (Fig. 13). Additionally, we investigated whether overlapping cpACP, which exhibits direct anticancer activity, could enhance anticancer activity. As a result, overlapping ACP, fusion of the N term of cpACP, and overlapping cpACP were tested, but no enhancement of anticancer activity was observed (Fig. 14). 5-2. Expression and purification of fusion proteins ACP and iRGD were deleted from the synthesized nucleic acid sequence through XbaI and SalI site deletion, and subcloned into the restriction enzyme sites SacI site and PstI site of pRK793 (Addgene; Plasmid #8827), an expression vector in which the MBP nucleic acid sequence is inserted, to produce [MBP-TEV cleavage sequence-scFv], [MBP-TEV cleavage sequence-scFv-ACP], and [MBP-TEV cleavage sequence-scFv-ACP-iRGD] recombinant expression vectors, respectively. The nucleic acid sequence of TEV protease was PCR-amplified from pRK793 using a forward primer (5'-AACGAGCTCGGGAGAAAGCTTG TTTAAGGGGCCG-3') and a reverse primer (5'-GTCGAGCTCGAGGAACCATTCATGAGTTGAGTCGCTTCCTTAACTGG-3') with restriction enzyme sites SacI site and XhoI site added, and this was subcloned into the SacI site of the recombinant expression vector to produce a vector for expression of a fusion protein having the structures [MBP-TEV-TEV cleavage sequence-scFv], [MBP-TEV-TEV cleavage sequence-scFv-ACP], and [MBP-TEV-TEV cleavage sequence-scFv-ACP-iRGD]. The above-mentioned fusion protein expression vector was transformed into E. coli BL21 (DE3) cells by heat shock at 42°C, plated on LB solid medium (Ampicillin 100 ug / mL), and cultured for 15 hours to select superior strains. The selected superior strains were then induced to express in 500 mL of LB liquid medium (Ampicillin 100 ug / mL) at 15°C for 16 hours at an OD600 = 1.0, IPTG 0.2 mM, and 15°C, and approximately 3 g of cells were obtained from the cultured culture. Thereafter, the obtained cells were suspended in PBS to disrupt the cells, and the supernatant was applied to a Ni-NTA column, the column was washed with 25 mM imidazole, and each fusion protein was eluted with 500 mM imidazole. E. coli transformed with the above fusion protein expression vector expressed fusion proteins of the structures [MBP-TEV-TEV cleavage sequence-scFv], [MBP-TEV-TEV cleavage sequence-scFv-ACP], and [MBP-TEV-TEV cleavage sequence-scFv-ACP-iRGD], which were cleaved in E. coli, and only [scFv], [scFv-ACP], and [scFv-ACP-iRGD] were purified, respectively. In short, [MBP-TEV-TEV cleavage sequence-scFv], [MBP-TEV-TEV cleavage sequence-scFv-ACP] and

[0124] Plasmids encoding fusion proteins of the structure [MBP-TEV-TEV cleavage sequence-scFv-ACP-iRGD] were expressed in E. coli (with or without IPTG) and purified by Ni-NTA chromatography using Ni-NTA resin, and the purified proteins were confirmed using SDS-PAGE (Fig. 15). Looking at Figure 15, [scFv], [scFv-ACP], and [scFv-ACP-iRGD] cleaved within the cell by TEV protease were present in the available fraction (S), and were purified at once by Ni-NTA chromatography or pull-down method. Meanwhile, when a plasmid encoding a protein produced by attaching only His6 to the scFv without the MBP-TEV -TEV cleavage sequence was expressed in E. coli, the protein was mostly expressed insoluble and existed in the pellet (P), and only a very small amount was expressed soluble. 5-3. Confirmation of binding affinity of fusion protein to target antigen The binding affinity of the fusion proteins scFv, scFv-ACP and scFv-ACP-iRGD expressed and purified in the above 5-2 to the target antigens (HER2 and SKOV3) was confirmed. Specifically, HER2 (Human Epidermal growth factor receptor2) protein (Acro biosystem, HE2-H5225) was diluted to 50 ng / mL in coating buffer (200 mM Na2Co3, 200 mM NaHCo3, pH 9.6), dispensed into 96-well ELISA plates at 100 μl each, and allowed to adsorb overnight at 4°C. Afterwards, the plates were washed three times with PBS-T (PBS, 0.05% Tween20) to remove unadsorbed antigen, and 200 μl of 10% skim milk (PBS, 5% w / v skim milk powder) was added to each well at room temperature, blocked for 1 hour, and then removed. 100 μl of scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in the above 5-2 were treated to each well at a concentration of 0.1 pM to 400 nM, respectively, and incubated at room temperature for 1 hour. Unbound antibodies were removed by washing three times with PBS-T (PBS, 0.05% Tween20), and 100 μl of ProteinL-HRP (Genscript, M00098) (in PBS) for detecting VL was treated to each well at a concentration of 50 ng / mL at room temperature, and incubated for 1 hour. Unbound ProteinL-HRP was removed by washing three times with PBS-T (PBS, 0.05% Tween20), and 100 μl of TMB containing 1.5% H2O2 was treated to each well at room temperature, and a color reaction was induced for 30 minutes. Afterwards, 100 μl of 1 M HCl was added to each well to stop the reaction, and the absorbance was measured at 450 nm. Each experiment was repeated three times, and the measured values ​​and standard deviation at each concentration were plotted using the Prism8 program. In addition, the specific binding affinity (KD value) was determined using ELISA and LSF (least-squares fit) (Fig. 16, left). In addition, ovarian cancer cell line SKOV-3 (HER2 overexpressing cell line) cultured in a culture dish at 37℃, 5% CO2 environment was washed and resuspended in 1% BSA (PBS, 1% Bovine Serum Albumin) and 10 6 Each well was dispensed into a 96-well U-type bottom plate. Then, 100 μl of scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in step 5-2 above were treated to each well at a concentration of 0.1 pM to 400 nM, and incubated at 4°C for 30 minutes. The cells were centrifuged at 4°C and 500 g for 5 minutes, resuspended in 1% BSA (PBS, 1% Bovine Serum Albumin), and washed twice with 1% BSA (PBS, 1% Bovine Serum Albumin). Afterwards, ProteinL FITC (Acrobiosystems, RPL-PF141) was diluted to 5 ug / mL in 1% BSA (PBS, 1% Bovine Serum Albumin) and treated with 100 μl per well and incubated in a darkroom at 4 °C for 30 min. After washing twice with 1% BSA (PBS, 1% Bovine Serum Albumin), the cells were centrifuged at 4 °C and 500 g for 5 min and resuspended in 100 μl of PBS. Afterwards, flow cytometry data were obtained using a FACSCalibur flow cytometer (BD, USA), and the measured values ​​and standard deviations were plotted using the Prism8 program. In addition, the specific binding affinity (KD value) was determined using flow cytometry and LSF (least-squares fit). (Figure 16, right). Looking at Figure 16, it can be confirmed that the fusion protein (scFv-ACP-iRGD) manufactured by sequentially linking an anticancer peptide and a cell-penetrating peptide to a tumor-targeting carrier has a higher binding affinity for target antigens (HER2 and SKOV3) than the tumor-targeting carrier alone (scFv) or the fusion protein (scFv-ACP) manufactured by linking an anticancer peptide to the tumor-targeting carrier. 5-4: Confirmation of the apoptotic ability of the fusion protein against cancer cells The cancer cell killing effects of scFv, scFv-ACP, and scFv-ACP-iRGD were investigated using SKOV3 (ovarian cancer cell line) and MDA-MB-231 (breast cancer cell line). Ovarian cancer cell line SKOV-3 (HER2 overexpressing cell line) was seeded at 4000 cells / well in 96-well plates with 90 μl of culture medium, and cultured for 4 hours at 37°C, 5% CO2. 10 μl of scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in 5-2 were added to each well plate, and treated once at concentrations of 0, 0.15, 0.31, 0.63, 1.3, 2.5, 5, and 10 μM, respectively, and cultured for 36 hours at 37°C, 5% CO2. After the culture was completed, 10 μl of CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) solution was added to the well plate and reacted for 2 hours at 37°C and 5% CO2 conditions. After the reaction was completed, the absorbance was measured using a microplate reader. Looking at Figure 17, it can be confirmed that the scFv, scFv-ACP, and scFv-ACP-iRGD of the present invention do not exhibit cell killing ability against cancer cells. These results suggest that when only the tumor-targeting delivery system (scFv) and the anticancer peptide are fused without the MMP cleavage sequence of the present invention, the cell killing ability may be rapidly reduced due to internalization and digestion in the endosome. Example 6: Fusion of a tumor-targeting delivery system, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide (scFv-MMP-ACP-iRGD) A fusion protein was manufactured by sequentially linking an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide to the tumor-targeting delivery system of the present invention, and then the cell killing ability of the fusion protein against cancer cells was confirmed. 6-1. Comparison of the apoptotic ability of fusion proteins against cancer cells according to the type of MMP cleavage sequence. A fusion protein was prepared by sequentially linking an MMP cleavage sequence (MA18, MA15), an anticancer peptide, and a cell-penetrating peptide to a tumor-targeting delivery system (see 5-1 above). The specific sequences of the components constituting each of the above fusion proteins are as shown in Table 4 below. Classification Tumor-targeting delivery system MMP cleavage sequence Anticancer peptide Cell-penetrating peptide D303 SEQ ID NO: 24 (scFv) SEQ ID NO: 25 (MA18) SEQ ID NO: 14 (ACP) SEQ ID NO: 16 (iRGD) D305 SEQ ID NO: 26 (MA15) After expressing and purifying the fusion protein manufactured above, it was confirmed through SDS-PAGE (see 5-2 above) (Fig. 18). Thereafter, the cell killing ability of fusion proteins (D303 and D305) manufactured by varying the MMP cleavage sequences against cancer cells was tested (see 5-4 above) (Fig. 19). Looking at Figure 19, it can be confirmed that both fusion proteins D303 and D305, which were manufactured with different MMP cleavage sequences, exhibit excellent cell killing ability against cancer cells (SKOV3 and MDA-MB-231). 6-2. Comparison of the cell killing ability of fusion proteins manufactured using different anticancer peptides and cell-penetrating peptides against cancer cells. A fusion protein was prepared by sequentially linking an MMP cleavage sequence, an anticancer peptide (ACP or KLAK), and a cell-penetrating peptide (mmdNP2, 9R, iRGD) to a tumor-targeting delivery system (see 5-1 above). The specific sequences of the components that make up each of the above fusion proteins are as shown in Table 5 below. Classification Tumor targeting carrier MMP cleavage sequence Anticancer peptide Cell penetrating peptide D301 SEQ ID NO: 24 (scFv) SEQ ID NO: 25 (MA18) SEQ ID NO: 15 (KLAK) SEQ ID NO: 19 (mmdNP2) D302 SEQ ID NO: 15 (KLAK) SEQ ID NO: 20 (9R) D303 SEQ ID NO: 14 (ACP) SEQ ID NO: 16 (iRGD) D304-- The fusion protein manufactured above was expressed and purified, and then confirmed through SDS-PAGE (see 5-2 above) (Fig. 20). Thereafter, the apoptotic ability of fusion proteins (D301, D302, D303, and D304) prepared by varying the anticancer peptide (ACP or KLAK) and cell penetrating peptide (mmdNP2, 9R, iRGD) against cancer cells was tested (see 5-4 above) (Fig. 21). Looking at Figure 21, it can be confirmed that the fusion proteins D301 and D303, which were manufactured by using different anticancer peptides and cell penetrating peptides, exhibit excellent cell killing abilities against cancer cells (SKOV3 and MDA-MB-231). The inventors of the present invention named the fusion protein (scFv / antibody-MMP-ACP-iRGD) of the tumor-targeting delivery system, MMP cleavage sequence, anticancer peptide, and cell-penetrating peptide, fabricated in Example 6, as the DTAT (Dual-Targeting Antibody-based Therapeutics) platform. This name was devised because the tumor is primarily targeted by the tumor-targeting delivery system, and the tumor is secondarily targeted by the cell-penetrating anticancer peptide targeting CP2c. Example 7: Fusion of serum albumin binding peptide, tumor-targeting delivery system, MMP cleavage sequence, anticancer peptide, and cell-penetrating peptide (saBP-scFv-MMP-ACP-iRGD) To confirm the possibility of fusion of saBP to increase the in vivo half-life of a dual-target antibody anticancer drug, a fusion protein was prepared by sequentially linking a serum albumin-binding peptide, a tumor-targeting delivery system, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide while varying the serum albumin-binding peptide. 7-1. Screening of serum albumin-binding peptides The specific sequences of the serum albumin binding peptides used in the experiments of the present invention are as shown in Table 6 below. Classification Amino acids SequenceSA20Ac-QRLIEDICLPRWGCLWEDDF-NH2SA21Ac-RLIEDICLPRWGCLWEDD-NH2SA22Ac-RLIEDICLPRWGCLWED-NH2S A29Ac-RLIEDICLPRWGCLWE-NH2SA31Ac-RLIEDICLPRWGCLW-NH2SA33Ac-RLIEDICLPRWGCL-NH2SA35Ac-RLIEDIC LPRWGC-NH2SA23Ac-LIEDICLPRWGCLWED-NH2SA24Ac-IEDICLPRWGCLWED-NH2SA25Ac-EDICLPRWGCLWED-NH2SA2 6Ac-DICLPRWGCLWED-NH2SA27Ac-ICLPRWGCLWED-NH2SA28Ac-CLPRWGCLWED-NH2SA30Ac-IEDICLPRWGCLWE-NH2 Through phage display screening of albumin-binding peptides (Mark S Dennis et. al, Albumin binding as a general strategy for improving the pharmacokinetics of proteins, J Biol Chem . 2002 Sep 20;277(38):35035-43.), we selected sequences with excellent half-maximal binding concentrations (IC50) as suggested in the literature, and identified sequences that specifically bind to albumin. In addition, the possibility of expression as expected hydrophilicity when applied to the dual-target anticancer drug platform was predicted and compared by expressing it as a hydrophilicity score, and the candidate sequences with excellent predicted values ​​are summarized in Table 7. Classification IC50 (nM)Hydrophilicity score SA20 2600.05 SA21 270±1100.18 SA22 430±1700.01 SA29 400±90-0.17 SA31 200-0.39 SA33 4,310±2,770-0.17 SA35 250,000-0.05 SA23 360±140-0.18 SA24 1,380±410-0.07 SA25 2,730±1,3000.06 SA26 3,120±660-0.17 SA27 86,700±21,800-0.43 SA28 400,000-0.31 SA30 1,800±590-0.29 In Table 7 above, serum albumin-binding peptides with an IC50 of 500 or higher were selected and used in the following tests. The specific sequences of the selected serum albumin-binding peptides are as shown in Table 8 below. Sequence number SA20 Sequence number 27 SA21 Sequence number 28 SA22 Sequence number 29 SA29 Sequence number 30 SA31 Sequence number 31 SA23 Sequence number 32 7-2. Preparation of fusion proteins with different serum albumin binding peptides A fusion protein was prepared by sequentially linking a serum albumin binding peptide, a tumor-targeting delivery system, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide while varying the serum albumin binding peptide (see 5-1 above). The specific sequences of the components constituting each of the above fusion proteins are as shown in Table 9 below. ClassificationSerum albumin binding peptideTumor targeting carrierMMP cleavage sequenceAnticancer peptideCell penetrating peptideD310-SEQ ID NO: 24 (scFv)SEQ ID NO: 25 (MA18)SEQ ID NO: 14 (ACP)SEQ ID NO: 16 (iRGD)D311SEQ ID NO: 27 (SA20)D312SEQ ID NO: 28 (SA21)D313SEQ ID NO: 29 (SA22)D314SEQ ID NO: 30 (SA29)D315SEQ ID NO: 31 (SA31)D316SEQ ID NO: 32 (SA23) The fusion protein manufactured above was expressed and purified, and then confirmed through SDS-PAGE (see 5-2 above) (Figs. 22 and 23). Looking at Figures 22 and 23, it can be confirmed that the expression and purification rates of the fusion proteins (D310, D311, D312, D314) that fuse SA20, SA21, and SA29 are more excellent. 7-3. Binding affinity of fusion proteins conjugated with saBP to target antigen The binding affinity of the fusion protein fused with SA20, SA21, and SA29 expressed and purified in the above 7-2 to the target antigens (HER2 and SKOV3) was confirmed (see 5-3 above) (Fig. 24). Looking at Figure 24, it can be confirmed that the fusion protein (D310, D311, D312, D314) manufactured by sequentially linking a tumor-targeting delivery system, an anticancer peptide, and a cell-penetrating peptide to saBP has excellent binding affinity for the target antigen and serum albumin. 7-4. Confirmation of the cell killing ability of the fusion protein combining saBP against cancer cells. The cell killing ability of the fusion protein fused with SA20, SA21, and SA29 expressed and purified in the above 7-2 against cancer cells was confirmed (see 5-4 above) (Fig. 25). Referring to Figure 25, the fusion proteins (D310, D311, D312, D314) manufactured by sequentially linking a tumor-targeting delivery system, an anti-cancer peptide, and a cell-penetrating peptide to the saBP of the present invention all exhibit excellent cell killing ability against cancer cells. Therefore, it can be seen that the fusion of saBP does not inhibit the cell killing ability of the fusion protein against cancer cells. Additionally, the anticancer activity of D311 was confirmed in various tumor cell lines, immune cell lines, and normal cell lines. As a result, D311 was found to induce cancer cell-specific apoptosis and exhibit anticancer effects ~10-fold greater than cisplatin. No apoptotic effects were observed in immune cells, normal cells, or stem cells (Fig. 26). In addition, CRO organization Qbestbio Co., Ltd. evaluated the nonclinical efficacy of DTAT D311 and DTAT D310 (w / o saBP) compared to Herceptin in SKOV3-implanted nude mice using the Q3Dx8 / IV scheme. The evaluation results showed that DTAT D311 was superior to Herceptin in tumor size reduction rate by 15.3% and tumor weight reduction rate by 16.9%. In addition, DTAT D311, which added the saBP sequence to increase the half-life, showed superior antitumor activity than DTAT D310 (w / o saBP) (Fig. 28). Example 8: DTAT platform containing mesothelin-targeting antibodies (DTAT-MSLN) In Example 6, DTAT-MSLN (Dual-Targeting Antibody-based Therapeutics for Mesothelin-positive Cancer) was developed using anti-mesothelin antibodies as tumor-targeting delivery vehicles on the DTAT platform (Figure 29). The anti-mesothelin antibodies used were 1T1G5, CSA0682 (developed by Partyabgen), and Anetumab (developed by Bayer), identified in Example 2. The sequences of CSA0682 and Anetumab are shown in Table 10 below. Sequence number 1T1G5 heavy chain sequence number 331T1G5 light chain sequence number 34CSA0682 heavy chain sequence number 35CSA0682 light chain sequence number 36Anetumab heavy chain sequence number 37Anetumab light chain sequence number 38 8-1. Comparison of the characteristics of three monoclonal anti-mesothelin antibodies Sequences encoding 1T1G5, CSA0682, and anetumab were cloned into expression vectors, expressed, isolated, and compared and analyzed for mass and relative affinity. Results showed that all three antibodies were well expressed and exhibited excellent affinity for mesothelin (Figures 30 and 31). 8-2. DTAT-MSLN Production Six dual-targeting anticancer agents were produced by applying the DTAT platform to three mesothelin antibodies. First, a light chain expression vector of mesothelin antibodies, a heavy chain expression vector, a fusion protein (L chain-MMP-ACP-iRGD) expression vector comprising a light chain-MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide, and a fusion protein (H chain-MMP-ACP-iRGD) expression vector comprising a heavy chain-MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide were produced (Figure 32). Afterwards, the corresponding expression vectors were co-transfected into CHO-s cells. At this time, when the DAR (drug antibody ratio) of DTAT-MSLN was 2:1, the heavy chain expression vector and the light chain-MMP-ACP-iRGD expression vector were co-transfected into the cells, and when the DAR was 4:1, the heavy chain-MMP-ACP-iRGD and light chain-MMP-ACP-iRGD expression vectors were used. Hereinafter, DTAT-MSLN with a DAR of 2:1 is referred to as DTAT2-MSLN, and DTAT-MSLN with a DAR of 4:1 is referred to as DTAT4-MSLN. In addition, DTAT-MSLN produced according to the mesothelin antibody used are referred to as follows: DTAT-1T1G5, DTAT-0682, and DTAT-Ane. Proteins expressed in cells were recovered and their productivity was compared. As a result, DTAT2-MSLNs were stably and repeatedly expressed well, and their productivity was similar to that of the monoclonal antibody. In contrast, DTAT4-MSLNs showed an expression rate similar to that of the monoclonal antibody, but only the cpACP of the heavy chain was expressed in a ~40% truncated form (Figures 33 and 34). The expression of the partially truncated cpACP of the heavy chain was determined to be due to the CS module cleavage enzyme expressed in CHO-S cells. 8-3. Comparison of binding affinities of DTAT-MSLN During maturation, mesothelin produces cell-surface mesothelin and megakaryocyte potentiating factor (MPF). Using mesothelin and MPF, we analyzed the binding affinity of three DTAT2-MSLNs to three mesothelin monoclonal antibodies by ELISA. Our analysis revealed that none of the three DTAT2-MSLNs binds to MPF, and the binding affinities of the DTATs compared to each monoclonal antibody did not significantly differ (Fig. 35). These results suggest that applying the DTAT platform to mesothelin monoclonal antibodies does not impede tumor targeting. Additionally, the binding affinities of three mesothelin monoclonal antibodies and three DTAT2-MSLNs to mesothelin were analyzed using ELISA and FACS. The analysis results showed that, although the affinity of DTAT2-MSLNs was somewhat reduced compared to the monoclonal antibodies, the absolute affinity values ​​indicated that this did not actually interfere with tumor targeting (Figs. 36 and 37). 8-4. Analysis of pro-cancer activity and cancer cell-specific cell death of DTAT-MSLN Nine cancer cell lines (Aspc-1, BxPC-3, Capan1, Capan2, MIA-Paca2, Panc1, SNU-231, SKOV3, and A431), an immune cell line (THP-1), and a normal cell line (CHOs) were seeded in plates at 25% confluency. Cells were treated with DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane at concentrations ranging from 0 to 10 μM and cultured for 24 h. Cell viability was assessed using the CCK-8 assay. As a result, it was found that cell death was induced by DTAT-MSLN in most cancer cells, but there was no difference in the survival rates of immune cell lines and general cell lines (Figs. 38 to 40). 8-5. Stability Evaluation of DTAT-MSLN The stability of three DTAT-MSLNs in buffers and serum was investigated. Specifically, DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane were added to buffer (0.1 M Tris-glycine, pH 7.6) or human serum, respectively, and stored at 4°C, 24°C, and 37°C. After a certain period of storage, samples were taken and degradation was examined by SDS-PAGE. As a result of the verification, it was found that DTAT2-0682 was stable for more than a month at all temperatures when stored in a buffer, DTAT2-1T1G5 showed a decomposition pattern from the 16th day at 24℃ and 37℃, and DTAT2-Ane showed a decomposition pattern from the 16th day at 24℃ (Fig. 41). In addition, when stored in human serum, DTAT2-0682 was stable for more than a month at 4°C and 37°C, and showed a pattern of degradation starting from the 32nd day at 24°C. DTAT2-1T1G5 was stable for more than a month at 4°C and 37°C, and showed a pattern of degradation starting from the 16th day at 24°C. DTAT2-Ane was stable for more than a month at 4°C, and showed a pattern of degradation starting from the 8th day at 24°C and the 16th day at 37°C (Fig. 42). In summary, the three DTAT-MSLNs are stable for more than one month when refrigerated in buffered form and for more than eight days when stored in serum at 37°C. Furthermore, the stability of three DTAT-MSLNs compared to Kadcyla was evaluated using ELISA and CCK-8 methods. Specifically, DTAT2-1T1G5, DTAT2-0682, and DTAT2-Ane were stored in buffer (0.1 M Tris-glycine, pH 7.6) or human serum, respectively. After 8 and 32 days of storage, samples were collected and analyzed for degradation by ELISA, or the collected samples were treated with cells and the IC50 was determined by CCK-8. Our results showed that DTAT2-MSLNs maintained an affinity of ~nM, although the affinity decreased slightly with increasing storage time in buffer and serum. Furthermore, both Kadcyla and DTAT2-MSLNs showed a slight decrease in cell death activity (~10%) with increasing storage time in buffer and serum conditions, and the stability of DTAT2-MSLNs was similar to that of Kadcyla (Figs. 43 and 44). 8-7. Nonclinical efficacy evaluation of three DTAT2-MSLNs Seven-week-old BALB / c nude mice (CRL form Japan) were randomly divided into five groups: G1-Mock (untreated, 6 mice); G2-DTAT2-An administration group (2.7 mg / kg, 8 mice); G3-DTAT2-1T1G5 administration group (2.7 mg / kg, 8 mice); G4-DTAT2-0682 administration group (2.7 mg / kg, 8 mice); and G5-Anetumab-ravtansine administration group (2.7 mg / kg, 4 mice). Each mouse was injected with AsPC-1 cells at a density of 1 × 10 7 Tumor formation was induced by inoculation with a mouse. When tumors reached 150 to 200 mm3, each test substance was administered subcutaneously once every three days for a total of eight doses. Tumor size was measured twice a week during administration, and at the end of the test, the mice were sacrificed, and the tumors were removed and weighed. When the degree of tumor growth inhibition was confirmed compared to G1 (Mock) 13 days after administration of the test substance, the three DTAT2-MSLNs inhibited tumor growth by 12-13% compared to the Mock and showed excellent anticancer activity of ~4.2% compared to Anetumab-ravtansine (Figure 45). 8-8. Confirmation of DTAT4-MSLNs production conditions To produce intact DTAT4-MSLNs, the CS (Cancer-Specific Cleavage Seq.) gene was knocked out in the expression cell line HEK293T. As a result, the CS_K.O.-HEK293T cell line was established, and the expression of intact DTAT4-0682 (DAR=4:1) was confirmed (Fig. 46). In addition, as a result of comparing the cell death efficiency of DTAT2-0682 produced by each cell line (CHO-S / 293T), there was no difference in anticancer efficacy, and as a result of comparing the cell death efficiency of DTAT4-0682 to that of DTAT2-0682, DTAT4-0682 was 27% superior (Fig. 47). Additionally, to produce intact DTAT4-MSLNs, experiments were conducted using CHO cells with different stabilizers and purification resins. Experimental results showed that Protein A, a commonly used whole-body purification resin, purified DTAT4-0682 in a cleaved form, whereas Protein L purified only intact DTAT4-0682. Protein G also purified only intact DTAT4-0682, but had a low purification yield (Fig. 48). Therefore, purification with Protein L is possible when the kappa chain is not the Lambda chain (in the case of DTAT4-Ane and DTAT4-1T1G5, the Lambda chain). Furthermore, when glycerol or Tween 20 was added during DTAT4-0682 expression, DTAT4-0682 was expressed only in its intact form, compared to when no stabilizer was added (w / o). However, the expression level decreased when Tween 20 was used. When the concentration of glycerol was varied, the truncated form decreased as the concentration increased, and the truncated form was not observed from 20% (Fig. 48). In summary, it was confirmed that DTAT4-MSLNs could be produced during expression by adding 20% ​​glycerol. 8-9. Confirmation of DTAT4-MSLNs characteristics Affinity analysis of six DTAT2 / 4-MSLNs compared to each monoclonal antibody was performed using ELISA and FACS. The affinity analysis results showed that while the affinity of DTAT2 / 4-MSLNs was somewhat reduced compared to the monoclonal antibodies, absolute affinity did not actually interfere with tumor targeting (Fig. 49 and Table 11). DTAT-1T1G5DTAT-0682DTAT-AnemABDTAT2DTAT4mABDTAT2DTAT4mABDTAT2DTAT4ELISA(MSLN)0.044 nM0.047 nM0.048 nM17.9 nM30.7 nM34.2 nM17.4 nM26.5 nM40.5 nMFlow cytometry(MIA PaCa-2 / Hmsln)0.6 nM1.2 nM2.2 nM2.6 nM6.1 nM7.9 nM5.5 nM6.5 nM7.9 nM In addition, the cell killing efficacy of common anticancer drugs, cisplatin, DTAT2-0682, and DTAT4-0682, was confirmed through comparison of IC50 and IC80 against AsPC1 cells. As a result, it was found that DTAT4-0682 (IC50=0.6) had a superior cell killing efficacy compared to DTAT2-0682 (IC50=1.06) (Fig. 50). Example 9: DTAT platform (DTAT2 / 4-Tra) containing HER2 targeting antibody 9-1. DTAT-Tra production DTAT2 / 4-Tra was produced using the same method as in Example 8-1, but using Trastuzumab, an antibody targeting HER2 (Human Epidermal Growth Factor Receptor 2 Protein), instead of the mesothelin-targeting antibody. DTAT4-Tra also had a problem with partial cleavage of the CS in the heavy chain, but a cell line clone in which the CS cleavage enzyme was knocked out was secured in the HEK293T expression cell line, producing the complete form of DTAT4-Tra. Furthermore, in the CHO expression cell line, the complete form of DTAT4-Tra was produced by adding glycerol during cell culture (Fig. 51). The molecular weight of the produced DTAT2 / 4-Tra was analyzed by MALDI TOF, and a single peak of the expected molecular weight was detected, indicating that DTAT2 / 4-Tra in its entirety was produced (Fig. 52A). The binding affinity of trastuzumab to the HER2 protein molecule, the target antigen, or to the HER2-overexpressing cell line SKOV3 was analyzed using ELISA and flow cytometry experiments. The analysis results confirmed that the application of the DTAT_platform original technology (CS-ACP-CPP) did not significantly reduce the affinity for the HER2 protein molecule or the HER2-overexpressing cell line SKOV3 (Fig. 52B). 9-2. Confirmation of anticancer activity of DTAT-Tra As a result of confirming the anticancer activity of the produced DTAT-Tra, it was found that the anticancer activity increased as the DAR ratio increased (Fig. 53). Furthermore, the anticancer activity of DTAT-Tra produced in various tumor cell lines, immune cell lines, and normal cell lines was confirmed. As a result, DTAT2 / 4-Tra was confirmed to induce cancer cell-specific apoptosis and exhibit anticancer effects ~10-fold greater than cisplatin. It did not exhibit apoptotic effects in immune cells, normal cells, or stem cells (Fig. 54). DTAT2-Tra, in which each motif representing the DTAT platform's core technology (CS-ACP-CPP) was replaced with a point mutant, was produced to determine the effect of each motif on anticancer activity. As a result, it was confirmed that cytotoxicity disappeared when even one of the motifs was replaced with a point mutant (Fig. 55). Considering the above results, the anticancer activity of DTAT-Tra is thought to be mediated by the following mechanisms (Figure 56): 1) Trastuzumab binds to HER2, targeting tumor cells; 2) cleavage of cpACP occurs; 3) cpACP enters tumor cells; and 4) cpACP targets the oncogene CP2c, leading to cell death. If even one of these processes is not fulfilled, cell death does not occur. Therefore, compared to existing antibody-drug conjugates (ADCs) that have toxicity issues toward non-tumor cells, DTAT-Tra exhibits excellent tumor specificity and safety against normal cells. Example 10: DTAT platform (DTAT2 / 4-Cet) containing EGFR targeting antibodies 10-1. DTAT-Cet production Using the same method as in Example 8-1, DTAT2 / 4-Cet was produced in CHO cells using Cetuximab, an antibody targeting Epidermal Growth Factor Receptor (EGFR), instead of the mesothelin-targeting antibody. A schematic diagram of the action of DTAT2 / 4-Cet is shown in Figure 57. However, the DTAT platform applied using the full cetuximab antibody encountered the problem of partial cleavage of CS in the heavy chain during expression (Figures 58a and 58b). However, in the CHO expression cell line, complete DTAT2 / 4-Cet was produced by adding 20% ​​glycerol during cell culture (Figure 58c). The production efficiency of DTAT2 / 4-Cet was confirmed, and it was confirmed that the production efficiency was not affected even when 20% glycerol was added during cell culture (Figs. 59a and 59b). In addition, the anticancer efficacy of DTAT2-Cet and DTAT4-Cet was confirmed in A231 cells, and it was confirmed that the anticancer activity increased as DAR increased (Fig. 59c). The molecular weight of the produced DTAT2-Cet was analyzed by MALDI-TOF, and a single peak with the expected molecular weight was detected, indicating that DTAT2-Cet was produced in its intact form. In contrast to ADCs, where antibody-drug binding is heterogeneous and the DAR does not decrease in an integer ratio, DTAT2-Cet showed a single peak, confirming that the DAR was constant (Fig. 60a). The relative affinity of DTAT2-Cet for EGFR1 protein or the EGFR1-overexpressing cell line A431 was analyzed using ELISA and FACS methods. The affinity analysis results confirmed that the affinity change of DTAT2-Cet was minimal compared to the monoclonal antibody (Figures 60b and 60c). These results indicate that the application of the DTAT_platform source technology (CS-ACP-CPP) hardly reduces the affinity for the EGFR1 protein molecule or the EGFR1-overexpressing cell line A431. 10-2. Confirmation of the anticancer activity of DTAT2 / 4-Cet The anticancer activity of the produced DTAT2 / 4-Cet was confirmed in various tumor cell lines, immune cell lines, and normal cell lines. As a result, we confirmed that DTAT2 / 4-Cet induces cancer cell-specific apoptosis and exhibits anticancer effects ~10-fold superior to cisplatin. No apoptotic effects were observed in immune cells, normal cells, or stem cells (Fig. 61). In addition, the anticancer activity increased in the order of DTAT-D351 (application of the DTAT platform to Cet scFv), DTAT2-Cet, and DTAT4-Cet, and it was confirmed that the anticancer activity increased as DAR increased. DTAT2-Cet, in which each motif representing the DTAT platform source technology (CS-ACP-CPP) was replaced with a point mutant, was produced to determine the effect of each motif on anticancer activity. As a result, it was confirmed that cytotoxicity disappeared when even one of the motifs was replaced with a point mutant (Fig. 62). Considering the above results, the anticancer activity of DTAT-Cet is mediated by the following mechanisms (Figure 63): 1) cetuximab binds to EGFR, targeting tumor cells; 2) cleavage of cpACP occurs; 3) cpACP enters tumor cells; and 4) cpACP targets the oncogenic gene CP2c, leading to cell death. If even one of these processes is not fulfilled, cell death does not occur. Therefore, compared to existing antibody-drug conjugates (ADCs) that have toxicity issues toward non-tumor cells, DTAT-Tra offers superior tumor specificity and safety against normal cells.

Claims

1. A heavy chain variable region comprising heavy chain CDR1 (complementarity determining region 1), CDR2 and CDR3, each represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and A light chain variable region comprising light chain CDR1, CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 57 and SEQ ID NO: 6, respectively; A mesothelin-specific antibody or an antigen-binding fragment thereof comprising:

2. In the first paragraph, the mesothelin-specific antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 4 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:

8.

3. In the first paragraph, the antigen-binding fragment is a mesothelin-specific antibody or an antigen-binding fragment thereof selected from the group consisting of Fab (fragment antigen binding), Fab', F(ab')2, Fv (variable fragment), dsFv (disulfice-stabilized Fv fragments), scFv (single-chain Fv), diabody, Fd, Fd', and BiTE (bispecific T-cell engager).

4. In the third paragraph, the scFv is a mesothelin-specific antibody or an antigen-binding fragment thereof, wherein a light chain variable region and a heavy chain variable region are linked by a peptide linker.

5. In the fourth paragraph, the peptide linker is a mesothelin-specific antibody or an antigen-binding fragment thereof using the sequence of SEQ ID NO: 11 (GGGSSRSSSSGGGGSGGGG).

6. A polynucleotide encoding a mesothelin-specific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

7. An expression vector comprising the polynucleotide of clause 6.

8. A non-human transformant into which the expression vector of Article 8 has been introduced.

9. A pharmaceutical composition for preventing or treating cancer comprising a mesothelin-specific antibody or an antigen-binding fragment thereof according to paragraph 1.

10. A pharmaceutical composition for preventing or treating cancer in claim 10, wherein the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, peritoneal cancer, endometrial cancer, stomach cancer, colon cancer, breast cancer, kidney cancer, thyroid cancer, prostate cancer, biliary tract cancer, cervical cancer, esophageal cancer, thymic cancer, and blood cancer.

11. Tumor targeting carrier; MMP cleavage site; anticancer peptide and cell penetrating peptide sequentially linked. The above tumor-targeting delivery agent is an antibody, or an antigen-binding fragment thereof, comprising an amino acid sequence that specifically binds to HER1, HER2, mesothelin or other tumor-associated antigen, A tumor-targeting antibody fusion protein, wherein the above MMP cleavage sequence includes an amino acid sequence that is cleaved by MMP (Matrix metalloproteinase).

12. A tumor-targeting antibody fusion protein according to claim 11, wherein the anti-HER1 antibody is Cetuximab and the anti-HER2 antibody is Trastuzumab.

13. In the 11th paragraph, the antibody comprising an amino acid sequence that specifically binds to mesothelin is a tumor-targeting antibody fusion protein characterized in that it is one selected from the group consisting of: a) a mesothelin-specific antibody according to claim 1; b) an antibody comprising a heavy chain represented by the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36; and c) An antibody comprising a heavy chain represented by the amino acid sequence of SEQ ID NO: 37 and a light chain comprising the amino acid sequence of SEQ ID NO:

38.

14. A tumor-targeting antibody fusion protein according to claim 1, characterized in that the antigen-binding fragment is any one selected from scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2.

15. A tumor-targeting antibody fusion protein according to claim 11, wherein the MMP cleavage sequence is represented by the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO:

26.

16. A tumor-targeting antibody fusion protein according to claim 11, wherein the anticancer peptide is represented by the amino acid sequence of SEQ ID NO: 14 or 15.

17. A tumor-targeting antibody fusion protein according to claim 11, characterized in that the anticancer peptide has a cell penetrating peptide (CPP) comprising any one amino acid sequence selected from SEQ ID NOS: 16 to 20 bound to the C-terminus.

18. An expression vector comprising a polynucleotide encoding a tumor-targeting antibody fusion protein of any one of claims 11 to 17.

19. An expression vector according to claim 18, characterized in that the expression vector expresses a second fusion protein comprising MBP (maltose-binding protein), TEV protease (Tobacco etch virus protease), a TEV protease cleavage sequence (cleavage site), and the tumor-targeting antibody fusion protein.

20. An expression vector according to claim 19, wherein the second fusion protein is characterized in that MBP, TEV protease, a TEV protease cleavage sequence, and a tumor-targeting antibody fusion protein are sequentially linked.

21. An expression vector according to claim 19, characterized in that the MBP comprises an amino acid sequence represented by sequence number 21.

22. An expression vector according to claim 19, characterized in that the TEV protease comprises an amino acid sequence represented by SEQ ID NO:

22.

23. An expression vector according to claim 18, characterized in that the TEV protease cleavage sequence comprises an amino acid sequence represented by SEQ ID NO:

23.

24. A host cell transformed with the expression vector of clause 17.

25. A pharmaceutical composition for preventing or treating cancer, comprising a tumor-targeting antibody fusion protein of any one of claims 11 to 17 as an active ingredient.

26. A method for treating cancer, comprising administering to a subject in need of treatment at least one selected from the group consisting of: A mesothelin-specific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5; A pharmaceutical composition for preventing or treating cancer according to claim 9; A tumor-targeting antibody fusion protein according to any one of claims 11 to 17; and A pharmaceutical composition for preventing or treating cancer according to claim 24.

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