Novel Anti-methothelin antibody and uses thereof

Abstract

The present invention relates to a novel anti-mesothelin antibody, a pharmaceutical composition for cancer treatment comprising the same, and a dual-target anticancer platform. The mesothelin antibody according to one example of the present invention has excellent mesothelin binding ability and can be usefully used for cancer treatment.

Description

Technology Field

[0001] The present invention relates to a novel anti-mesothelin antibody and a pharmaceutical composition for treating cancer containing the same. Background Technology

[0002] With the advancement of medicine, various anticancer drugs have been developed, broadly categorized into cytotoxic drugs, targeted drugs, and immunotherapy drugs. Cytotoxic drugs are medications that kill cancer cells through toxicity, while immunotherapy drugs kill cancer cells by strengthening the immune system to attack them. Targeted drugs are medications that act by targeting specific proteins within cancer cells. Unlike normal cells, cancer cells grow rapidly, but in some cases, their growth is amplified by signals from specific proteins. Targeted drugs identify and target these signals from cancer cells to inhibit their growth and proliferation.

[0003] Mesothelin (MSLN) is a cell surface glycoprotein that is normally expressed in serous tissues such as the pleura, pericardium, and peritoneum. It is not expressed in life-sustaining organs and is commonly overexpressed in many solid tumors, such as mesothelioma, pancreatic ductal adenocarcinoma, and ovarian cancer (Non-patent Literature 1), so it can be an ideal target for cancer treatment.

[0004] In addition, mesothelin has recently been attracting attention as a biomarker for pancreatic cancer (Non-patent Literature 2). Prior art literature

[0005] 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). The problem to be solved

[0006] Under the circumstances described above, the inventors made diligent efforts to produce a novel antibody that specifically binds to human mesothelin, and as a result, invented a novel antibody having a high affinity for mesothelin overexpressed in cancer cells, thereby completing the present invention. In addition, the inventors developed an anticancer platform comprising the above-mentioned anti-mesothelin antibody.

[0007] Accordingly, one objective of the present invention is to provide a novel anti-mesothelin antibody.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for treating cancer comprising the anti-mesothelin antibody or the anticancer platform. means of solving the problem

[0009] To achieve the above objective, one aspect of the present invention is

[0010] A heavy chain variable region comprising heavy chain CDR1 (complementarity determining region 1), CDR2, and CDR3, respectively represented by the amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; and

[0011] A mesothelin-specific antibody or its antigen-binding fragment is provided, comprising a light chain variable region including light chains CDR1, CDR2, and CDR3, respectively represented by the amino acid sequences of SEQ ID NO. 5, SEQ ID NO. 57, and SEQ ID NO. 6.

[0012] The 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 completed the present invention by identifying the sequences and complementarity determining sites (CDRs) of scFv expressed in the clones.

[0013] In the present invention, the term "antibody" refers to a protein molecule that acts as a receptor for specifically recognizing an antigen, comprising an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, and complete antibodies. Additionally, the term includes chimeric antibodies, humanized antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabadies, triabadies, and tetraabadies.

[0014] In the present invention, the term “antigen binding fragment” refers to a fragment having 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.

[0015] Among the antigen-binding fragments mentioned above, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. 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. The F(ab')2 antibody is produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds.

[0016] Fv is the smallest antibody fragment containing only the heavy chain variable region (VH) and the light chain variable region (VL), while in two-chain Fv, the heavy chain variable region and the light chain variable region are connected by non-covalent bonds. Single-chain variable fragments (scFv) are recombinant antibody fragments reconstructed by linking only the variable regions constituting the antigen-binding site from the light chain and heavy chain using a linker. Antibodies are large molecules of approximately 150 kDa, making it difficult for them to act effectively in large tumors or tissues that are difficult to penetrate. However, scFv is a molecule of approximately 30 kDa; because it is the smallest antibody fragment that maintains a complete antigen-binding site, it retains the antibody's inherent specific antigen-binding ability and can act effectively even in large tumors or tissues that are difficult to penetrate.

[0017] Fd is an antibody fragment composed of the variable region (VH) and the first constant region (CH1) of the heavy chain, and BiTE (bispecific T-cell engager) refers to an antibody designed to bind simultaneously to a specific antigen and a T-cell surface protein; for example, it may consist of two scFv molecules that recognize the surface antigen of a cancer cell and CD3 of a T cell, respectively.

[0018] The above antigen-binding fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restricting the whole antibody with papain and F(ab')2 fragment can be obtained by cleaving it with pepsin), and can be produced through genetic recombination technology.

[0019] In the present invention, the term "CDR (complementarity determining region)" refers to the amino acid sequence of a hypervariable region in the heavy chain and light chain of an antibody. The heavy chain and light chain may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide major contact residues for the antibody to bind to an antigen or epitope.

[0020] According to one embodiment 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.

[0021] 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 the sequence of SEQ ID NO. 11 (GGGSSRSSSSGGGGSGGGG) may be used as the peptide linker.

[0022] According to one embodiment of the present invention, the human mesothelin-specific scFv may include six histidine tags (6x His-tag) and an HA tag at the C-terminus for purification and tracking (Fig. 3).

[0023] 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 the binding affinity with an antigen, and the smaller the dissociation constant, the greater the binding affinity between the antibody and the antigen. The binding affinity can be determined by conventional methods.

[0025] Another aspect of the present invention provides a polynucleotide encoding the human mesothelin-specific antibody or its antigen-binding fragment, an expression vector comprising the polynucleotide, and a transformant into which the expression vector has been introduced.

[0026] According to one embodiment of the present invention, the polynucleotide coating the human mesothelin-specific scFv may include the nucleic acid sequence represented by SEQ ID NO. 8, and the polynucleotide encoding the human mesothelin-specific antibody may include the nucleic acid sequences represented by SEQ ID NOs. 9 and 10, but is not limited thereto.

[0027] Meanwhile, a vector containing a polynucleotide encoding a human mesothelin-specific antibody may include a polynucleotide encoding either the heavy chain or the light chain of the antibody, respectively, or may include a polynucleotide encoding both the heavy chain and the light chain. If the polynucleotides encoding the heavy chain or the light chain of the antibody exist in separate vectors, the heavy chain-coding vector and the light chain-coating vector may be introduced into a single transformant or into separate transformants. If the vectors are introduced into separate transformants, the expressed antibody light chain and antibody heavy chain can be recovered to produce an intact antibody.

[0028] In the present invention, an expression vector comprising a polynucleotide encoding the human mesothelin-specific antibody or its antigen-binding fragment may be a vector capable of replicating and / or expressing said polynucleotide in prokaryotic or eukaryotic cells, including bacterial cells (e.g., E. coli, etc.), mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, or insect cells, although it is not particularly limited thereto. Preferably, it may be a vector comprising at least one selection marker that is operably linked to a suitable promoter so that said polynucleotide can be expressed in a host cell. Examples include a form in which said polynucleotide is introduced into a phagemid, plasmid, cosmid, mini-chromosome, viral or retroviral vector, etc.

[0029] In the present invention, the transformant into which the expression vector is introduced is not particularly limited thereto, but may be a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella typhimurium into which the expression vector is introduced and transformed; a yeast cell; a fungal cell such as Pichia pastoris; an insect cell such as Drozophylla or Spodoptera Sf9 cell; 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.

[0030] The delivery of the above polynucleotide or an expression vector containing it into a host cell / transformer may be carried out using a delivery method widely known in the industry. For example, if the host cell is a prokaryotic cell, the delivery method may be the CaCl2 method or the electroporation method, and if the host cell is a eukaryotic cell, the delivery method may be the microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, and gene bombardment method, but is not limited thereto.

[0032] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the mesothelin-specific antibody or its antigen-binding fragment as an active ingredient.

[0033] Mesothelin is low in normal tissues, but overexpression has been confirmed in various types of solid tumors, including mesothelial carcinoma, pancreatic cancer, and ovarian cancer, and is being studied as an anticancer target. For example, Korean registered patent No. 10-2070016 discloses a chimeric antigen receptor containing a binding domain that specifically binds to mesothelin and the anticancer effect of T cells expressing it. Therefore, mesothelin-specific antibodies can be usefully employed for anticancer purposes.

[0034] The above cancer may be selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, peritoneal cancer, endometrial cancer, stomach cancer, colorectal cancer, breast cancer, kidney cancer, thyroid cancer, prostate cancer, cholangiocarcinoma, cervical cancer, esophageal cancer, thymic cancer, and hematological cancer.

[0035] In addition to including a mesothelin-specific antibody or its antigen-binding fragment as an active ingredient, the above pharmaceutical composition may further include a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceuticals.

[0036] The pharmaceutical composition according to the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, each according to conventional methods. Carriers, excipients, and diluents that may 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, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0037] When formulating, the formulation is prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules, and these solid formulations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition of the present invention. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerol gelatin, etc. may be used.

[0038] The composition of the present invention may be administered orally or parenterally, and any parenteral administration method may be used. Systemic or local administration is possible, but systemic administration is more preferable, and intravenous administration is most preferable.

[0039] 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 form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. Administration may be performed once a day or divided into several doses, and the said dosage does not limit the scope of the present invention in any way.

[0041] 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 connected, wherein the MMP cleavage site comprises an amino acid sequence that is cleaved by a matrix metalloproteinase (MMP).

[0042] According to one embodiment of the present invention, the antibody that specifically binds to mesothelin may be selected from the group consisting of the following, but any other antibodies known as mesothelin-specific antibodies in the technical field to which the present invention belongs may be used.

[0043] a) an antibody (1T1G5) comprising a heavy chain represented by the amino acid sequence of SEQ ID NO. 33 and a light chain formed by the amino acid sequence of SEQ ID NO. 34; and b) an antibody (Anetumab) comprising a heavy chain represented by the amino acid sequence of SEQ ID NO. 37 and a light chain formed by the amino acid sequence of SEQ ID NO. 38.

[0044] delete

[0045] delete

[0046] According to one embodiment 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.

[0047] According to one embodiment of the present invention, the tumor-targeting antibody fusion protein primarily targets a tumor with a mesothelin antibody and induces tumor cell death with an anticancer peptide. To facilitate the influx of the anticancer peptide into the cell, the anticancer peptide may have a cell-penetrating peptide attached to its C-terminus comprising any one amino acid sequence selected from SEQ ID NOs 16 to 20.

[0049] Another aspect of the present invention provides an expression vector comprising a polynucleotide encoding a tumor-targeting antibody fusion protein.

[0050] According to one embodiment of the present invention, the expression vector may express a second fusion protein comprising MBP (maltose-binding protein), TEV protease (Tobacco etch virus protease), TEV protease cleavage site, and the tumor-targeting antibody fusion protein.

[0051] Specifically, the second fusion protein may be one in which MBP, TEV protease, TEV protease cleavage sequence, and tumor-targeting antibody fusion protein are sequentially linked.

[0052] According to one embodiment of the present invention, the MBP may include an amino acid sequence represented by SEQ ID NO. 21, and the TEV protease may include an amino acid sequence represented by SEQ ID NO. 22. Additionally, the TEV protease cleavage sequence may include an amino acid sequence represented by SEQ ID NO. 23.

[0053] Generally, when scFv (or tumor-targeting antibody fusion proteins containing scFv) is expressed in E. coli, most of them are expressed in an insoluble manner, which presents a problem in that the amount of functionally usable protein expression is very small. Accordingly, conventionally, attempts have been made to improve the production yield of scFv (or tumor-targeting antibody fusion proteins containing scFv) in E. coli by fusing the pelB sequence, but these have not been very effective. Although fusing MBP was efficient for solubilizing scFv (or tumor-targeting antibody fusion proteins containing scFv), separating pure scFv (or tumor-targeting antibody fusion proteins containing scFv) from the fusion protein required an additional purification process, which was cumbersome and time-consuming.

[0054] Accordingly, the inventors have devised an expression system capable of purifying soluble scFv (or a tumor-targeting antibody fusion protein containing scFv) in E. coli in a single step without additional processes. According to the expression vector of the present invention and the method for producing a protein using said expression vector, the target protein can be produced homogeneously in a soluble form within E. coli, and since the target protein is cleaved by the TEV protease expressed within the cell, there is an effect of purifying only the pure target protein in one step.

[0055] Another aspect of the present invention provides a tumor-targeting antibody fusion protein comprising a whole antibody; an MMP cleavage site; an anticancer peptide and a cell-penetrating peptide sequentially connected, wherein the MMP cleavage site comprises an amino acid sequence that is cleaved by a matrix metalloproteinase (MMP).

[0056] In the present invention, the whole antibody refers to an antibody composed of a light chain and a heavy chain that includes both a variable region and a constant region.

[0057] According to one embodiment of the present invention, the total antibody includes, but is not limited to, a mesothelin antibody, a HER2 target antibody and an EGFR target antibody.

[0058] According to one embodiment of the present invention, the HER2 target antibody may be trastuzumab comprising a heavy chain represented by SEQ ID NO. 49 and a light chain represented by SEQ ID NO. 50.

[0059] In addition, the EGFR target antibody may be cetuximab comprising a heavy chain represented by SEQ ID NO. 53 and a light chain represented by SEQ ID NO. 54.

[0061] Another aspect of the present invention provides a host cell transformed with an expression vector of the tumor-targeting antibody fusion protein. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used.

[0063] Another aspect of the present invention provides a pharmaceutical composition for cancer treatment comprising the tumor-targeting antibody fusion protein, wherein all contents described in relation to the pharmaceutical composition comprising the mesothelin antibody may be applied or mutatis mutandis to the pharmaceutical composition for cancer treatment comprising the tumor-targeting antibody fusion protein. Effects of the invention

[0064] An antibody according to one example of the present invention has excellent binding ability to human mesothelin, so it can be used for the treatment of various cancers, including pancreatic cancer, and in dual-target anticancer platforms. Brief explanation of the drawing

[0065] Figure 1 shows the results of identifying phage clones binding to human mesothelin using a scFv phage library. Figure 2 shows the results of identifying phage clones binding to human mesothelin by ELISA screening. 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 (named mAb-1T1G5) that specifically binds to human mesothelin. Figure 5 shows the results of identifying scFv (named 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 confirming the mesothelin-specific binding ability of 1T1G5 by ELISA. Figure 7 is mesothelin Figure 8 shows the results of confirming the fluorescence signal of the antibody after reacting positive cell lines with 1T1G5 at different concentrations. 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 testing the apoptotic ability of a fusion protein obtained by fusing various peptides (iRGD, dNP2, mdNP2, mmdNP2, or 9R) expected to have cell permeability ability to the anticancer peptide (ACP or KLAK) of the present invention. Figure 10a is the result of testing the apoptotic ability of the fusion protein against cancer cells, and Figure 10b is a graph showing the numerical apoptotic ability of the fusion protein against cancer cells. Figure 11 is the result of further testing the apoptotic ability of a fusion protein by fusing various peptides (TAT, Amtd, Pep1, dNP2, 9R, and iRGD) expected to have cell penetration ability to an anticancer peptide (ACP or KLAK).Figure 12 is a schematic diagram showing a tumor-targeting antibody fusion protein formed by fusing a tumor-targeting carrier, an anticancer peptide, and a cell-penetrating peptide while omitting the MMP cleavage sequence. 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 the Cathapsin B cleavage site. Figure 14 shows the results of confirming the in vitro anticancer activity of a fusion protein by superimposing cpACP, which exhibits anticancer activity. Figure 15 shows the results of confirming the expression of a tumor-targeting antibody fusion protein formed by fusing a tumor-targeting carrier, an anticancer peptide, and a cell-penetrating peptide while omitting the MMP cleavage sequence using SDS-PAGE. Figure 16 is a graph showing the binding affinity of a tumor-targeting antibody fusion protein formed by fusing a tumor-targeting carrier, an anticancer peptide, and a cell-penetrating peptide while omitting the MMP cleavage sequence to the target antigen, and Figure 17 shows, with the MMP cleavage sequence omitted, These are results showing the apoptotic effect on cancer cells of a tumor-targeting antibody fusion protein fused with a tumor-targeting delivery vehicle, an anticancer peptide, and a cell-penetrating peptide. Figure 18 is a figure confirming the tumor-targeting antibody fusion proteins (D303 and D305) fused with a tumor-targeting delivery vehicle, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide by SDS-PAGE. Figure 19 is a figure confirming the apoptotic effect of the fusion proteins (D303 and D305) on cancer cells. Figure 20 is a figure confirming the fusion proteins (D301, D302, D303, and D304) by SDS-PAGE. Figure 21 is a figure confirming the apoptotic effect of the fusion proteins (D301, D302, D303, and D304) on cancer cells.Figure 22 shows the results of confirming the expression of tumor-targeting antibody fusion proteins (D310, D311, D312, D313, D314, D315, D316) fused with a tumor-targeting delivery vehicle, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide by SDS-PAGE: 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 (protein not bound to resin); and E-elution (protein eluted from resin). Figure 23 shows the results of confirming the expression of tumor-targeting antibody fusion proteins (D310, D311, D312, and D314) fused with a tumor-targeting delivery vehicle, an MMP cleavage sequence, an anticancer peptide, and a cell-permeable peptide by SDS-PAGE. Figure 24 shows the results of the analysis of the specificity and affinity of the fusion proteins (D310, D311, D312, and D314) for HER2 antigen or HSA (serum albumin) protein, and Figure 25 shows the results showing the apoptotic effect of the fusion proteins (D310, D311, D312, and D314) on cancer cells. Figure 26 shows the results confirming the anticancer activity of D311 in various tumor cell lines, immune cell lines, and normal cell lines. Figure 27 shows SKOV3-transplanted nude mice with the fusion protein (D310 or This is the result of confirming tumor growth after administering D311). Fig. 28 is a figure showing the tumor growth curve over time in an SKOV3 transplanted mouse model after administering fusion proteins (D310, D311) according to one embodiment of the present invention. Fig. 29 is a schematic diagram showing the operation of the DTAT-MSLN platform using an anti-mesothelin antibody. Fig. 30 shows the expression vector (A), expression result (B), and mass spectrometry result (C) of three anti-mesothelin antibodies 1T1G5 and Anetumab (Ane).Figure 31 shows the results of confirming the affinity of three anti-mesothelin antibodies, 1T1G5 and anetumab (Ane), for mesothelin using ELISA and flow cytometry. 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 DTAT-MSLN types (DTAT-1T1G5 and DTAT-Ane). Figure 34 shows the results of confirming the expression levels of three DTAT-MSLN types (DTAT-1T1G5 and DTAT-Ane). Figure 35 shows the results of confirming the affinity of three DTAT-MSLN types (DTAT-1T1G5 and DTAT-Ane) for mesothelin (MSLN) and megakaryocyte-stimulating factor (MPF). Figure 36 shows three DTAT-MSLN types This is the result of comparing the affinity of (DTAT-1T1G5 and DTAT-Ane) for mesothelin (MSLN) with anti-mesothelin antibodies. Figure 37 shows the results of confirming the affinity of three types of DTAT-MSLN (DTAT-1T1G5 and DTAT-Ane) for mesothelin (MSLN) using ELISA and FACS. Figure 38 shows the results of confirming the degree of apoptosis using IC50 after treating cancer cell lines (Aspc-1, BxPC-3, Capan1, Capan2, MIA-Paca2, Panc1, SNU-231, SKOV3, and A431), immune system cell lines (THP-1), and normal cell lines (CHOs) with DTAT2-1T1G5. Figure 40 shows cancer cell lines (Aspc-1, BxPC-3, Capan1, Capan2, MIA-Paca2, Figure 41 shows the results of confirming the degree of cell death as IC50 after treating Panc1, SNU-231, SKOV3, and A431), immune system cell lines (THP-1), and general cell lines (CHOs) with DTAT2-Ane. Figure 41 shows the results of confirming the degree of degradation after storing three types of DTAT-MSLN (DTAT-1T1G5 and DTAT-Ane) in buffer (0.1 M Tris-glycine, pH 7.6).Figure 42 shows the results of confirming the degree of degradation of the three DTAT-MSLN types (DTAT-1T1G5 and DTAT-Ane) after storage in human serum. Figure 43 shows the results of confirming the degree of degradation of the three DTAT-MSLN types (DTAT-1T1G5 and DTAT-Ane) by ELISA after storage in buffer (0.1 M Tris-glycine, pH 7.6). Figure 44 shows the results of confirming the degree of degradation of the three DTAT-MSLN types (DTAT-1T1G5 and DTAT-Ane) after storage in human serum by ELISA. Figure 45 shows the results of confirming the effects after administering the three DTAT-MSLN types (DTAT-1T1G5 and DTAT-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 49 shows the results of analyzing the affinity of six types of DTAT2 / 4-MSLNs against each monoantibody: A-ELISA; and B-analyzed by FACS. 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 DTAT-Tra expression while increasing glycerol concentration; and F-Results of DTAT-Tra expression under 20% glycerol conditions. Figure 52 shows the results of characterizing DTAT-Tra: A-mass spectrometry results; B-affinity confirmation results for HER2; Results of confirming affinity for C-SKOV3 cells. Fig. 53 shows the results of confirming the anticancer activity of DTAT2-Tra and DTAT4-Tra. Fig. 54 shows the results of confirming anticancer activity after treating cancer cell lines, immune system cell lines, and normal cell lines with DTAT2-Tra and DTAT4-Tra. Fig. 55 shows the results of inducing mutations in each motif constituting DTAT2-Tra (A), confirming the expression of the mutants (B), and confirming the anticancer activity of the mutants (C).Figure 56 is a schematic diagram illustrating the principle by which DTAT-Tra exhibits anticancer activity. Figure 57 is a schematic diagram illustrating 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 results; C - Results of expressing DTAT-Cet while increasing glycerol concentration. Figure 59 shows the production results and comparison of anticancer effects of DTAT-Cet: A - Results of expression with added 20% glycerol; B - Results of production efficiency confirmation when expressed with added 20% glycerol; C - Results of comparison of anticancer activity between DTAT2-Cet and DTAT4-Cet. Figure 60 shows the results of characterizing DTAT-Cet: A - Mass spectrometry results; B - Results of affinity confirmation for EGFR1; and results of confirming affinity for A431 cells. Fig. 61 shows the results of confirming anticancer activity after treating cancer cell lines, immune system cell lines, and normal cell lines with DTAT2-Cet and DTAT4-Cet. Fig. 62 shows the results of inducing mutations in each motif constituting DTAT2-Cet (A), confirming the expression of the mutants (B), and confirming the anticancer activity of the mutants (C). Fig. 63 is a schematic diagram showing the principle by which DTAT-Cet exhibits anticancer activity. Specific details for implementing the invention

[0066] One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments.

[0068] Example 1: Human Anti-Mesothelin Antibody Screening

[0069] 1-1. Phage Display

[0070] An M13 bacteriophage library was constructed in which variable regions (heavy and light chain variable regions) of various antibodies were expressed on the surface in the form of single-chain variable fragments (scFvs). Subsequently, antibodies were selected through a panning process. Panning is a process of selecting antibodies that bind to a specific antigen from a pool of various antibodies; a pool of antibody candidates that selectively bind to mesothelin was selected through five panning cycles. The detailed process is as follows.

[0071] 1) Mesothelin antigen was coated onto an immune tube at a concentration of 1 µg / ml (coating solution: sodium bicarbonate buffer (pH 9.6)).

[0072] 2) The tube was washed with PBS (PBST) containing 0.05% (v / v) Tween 20 to remove unbound mesothelin.

[0073] 3) The tube was blocked with 3% skim milk.

[0074] 4) A 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 the phage to mesothelin.

[0075] 5) The tube was washed to remove phages that were not bound to mesothelin.

[0076] 6) Phages showing affinity for mesothelin were isolated.

[0077] 7) The isolated phages were amplified by infecting host cells or bacteria.

[0078] 8) Return to Step 1) and repeat Steps 2) and 3) 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.

[0079] The results of the grip display are shown in Figure 1.

[0081] 1-2. ELISA Screening

[0082] ELISA screening was performed to select antibodies that specifically bind to mesothelin in independent clonal units.

[0083] Phages selected in Example 1-1 were added to plates coated with or uncoated with mesothelin antigen, and absorbance was measured at OD450. Phages were selected for which the difference between the absorbance measured on the mesothelin-coated plate and the absorbance measured on the mesothelin-coated plate was 1 or greater.

[0084] Screening results confirmed two phages that specifically bind to mesothelin (Fig. 2). Additionally, sequencing results confirmed that the scFv expressing the two phages is a single clone.

[0085] In Figure 2, D31 is the absorbance measured on the plate coated with the antigen, Background control (BC) is the absorbance measured on the plate not coated with the antigen, and delta is the difference in absorbance between the two plates.

[0087] Example 2: Preparation of antibody expression vector

[0088] 2-1. Production of Anti-Mesothelin Antibody Expression Vector

[0089] The antibody sequences confirmed in Examples 1-2 are listed in Table 1.

[0090] order number explanation amino acid sequence 1 HCDR1 GFTFSSYA 2 HCDR2 ISGSGGST 3 HCDR3 AKRVITLDY 4 variable heavy (VH) chain fragment QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRVITLDYWGQGTLVTVSS 5 LCDR1 SSNIGSNY 57 LCDR2 RNN 6 LCDR3 GTWDSSLSAVV 7 variable light (VL) chain fragment LTQPPSVSGSPGQSITISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDSSLSAVVFGGGTQLTVL

[0092] The sequences of 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 light chain and heavy chain sequences of the monoclonal antibody were expressed in separate vectors.

[0094] 2-2. Antibody Expression and Purification

[0095] - scFv production and purification

[0096] The vector prepared in Example 2-1 was transformed into Escherichia coli BL21(DE3) by the heat shock method. The transformed BL21(DE3) was plated on LB agar medium containing ampicillin and cultured at 37°C for at least 16 hours.

[0097] One colony of BL21(DE3) was inoculated into LB medium containing ampicillin and cultured until the OD 600 nm reached 1.0. Subsequently, 1 mM IPTG was added to induce protein expression, and the colony was cultured for an additional 16 hours.

[0098] After the culture was finished, the culture medium was centrifuged to recover only the cells, and the cells were lysed by sonication. The cell lysate was centrifuged to recover only the supernatant. Affinity chromatography was performed on the supernatant using Ni-NTA agar resin to purify scFv (hereinafter referred to as 1T1G5).

[0100] - mAb-1T1G5 production and purification

[0101] The vector of Fig. 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 a humidity of 80% or higher. After 5 days, the culture medium was centrifuged to collect the supernatant. The supernatant was passed through Protein A resin to perform affinity chromatography, and the anti-mesothelin monoclonal antibody (hereinafter referred to as mAb-1T1G5) was purified.

[0103] - Antibody purification results

[0104] The sizes of the purified 1T1G5 and mAb-1T1G5 were determined. The estimated sizes of 1T1G5 and mAb-1T1G5 are 26.3 kDa and 143.5 kDa, respectively. In addition, the estimated sizes of mAb-1T1G5 HC (heavy chain) and mAb-1T1G5 LC (light chain) are 48.9 kDa and 22.8 kDa, respectively.

[0105] Upon verification, it was confirmed that both 1T1G5 and mAb-1T1G5 had the expected size (Fig. 5).

[0107] Example 3: Confirmation of the antibody's mesothelin binding ability

[0108] 3-1. ELISA (Enzyme-Linked Immunosorbent Assay)

[0109] Mesothelin, the antigen, and HSA (human serum albumin), the negative control, were added to 24 wells at a dose of 50 ng per well and coated at 4°C for 16 hours. Bicarbonate / carbonate coating buffer (100 mM, pH 9.6) was used as the coating buffer. Subsequently, the coating buffer and antigen solution were removed, and each well was washed three times with 100 µl of PBST (PBS + 0.05% (v / v) Tween 20). After treating each well with 200 µl of blocking buffer (PBS + 1% (w / v) BSA), the plates were stored at room temperature for 1 hour. After removing the blocking buffer, 1T1G5, serially diluted with PBS in 24 steps from 500 nM to 0 nM in ½ increments, was treated into each well, and the plates were stored at room temperature for 1 hour. The PBS+1T1G5 solution was removed, and each well was washed 4 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; the plate was then stored at room temperature for 1 hour. The PBS+secondary antibody solution was removed, and each well was washed 4 times with 100 µl of PBST+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. Absorbance was measured at 450 nm.

[0110] Measurement results showed that 1T1G5 is a mesothelin-specific antibody that does not bind to HSA but binds only to mesothelin. In particular, the Kd value of 1T1G5 was calculated to be 11.4 nM, confirming its excellent binding affinity (Fig. 6).

[0113] 3-2. Flow cytometry

[0114] - Experimental group

[0115] 1T1G5 conjugated to the MSLN+ cell line MIA PaCa-2 / hMSLN

[0116] 2. MSLN+ cell line MIA PaCa-2 / hMSLN conjugated with mAb-1T1G5

[0117] - Experimental method

[0118] Mesothelin-positive cell line MIA PaCa-2 / hMSLN cells were prepared in FACS buffer (PBS + 2% FBS). 1T1G5 and mAb-1T1G5 were prepared by serial dilution, and 10^6 cells were treated at each concentration for 1 hour at 4°C. Afterward, the cells were harvested by centrifugation at 500g at 4°C for 3 minutes, and then washed three times with 100 µl of PBS.

[0119] Anti-HA-tag-Dylight 650, the secondary antibody for 1T1G5, was diluted 1:3000 in PBS, and ProteinA-FITC, the secondary antibody for mAb-1T1G5, was diluted 1:1000 in PBS. 100 µl of the diluted secondary antibodies were applied to each cell, and the cells were stored at 4°C for 1 hour. Cells were harvested by centrifugation at 500g at 4°C for 3 minutes, and then washed four times with 100 µl of PBS. The cells were resuspended in 300 µl of FACS buffer, and then analyzed using a flow cytometry instrument (1T1G5: 650 / 660 nm; mAb-1T1G5: 495 / 519 nm).

[0120] Analysis results confirmed that both 1T1G5 and mAb-1T1G5 bind to MSLN-positive cell lines (Figs. 7 and 8).

[0122] Example 4: Fusion of anticancer peptide and cell-permeable peptide

[0123] An anticancer peptide with tumor cell killing ability was fused with a cell-penetrating peptide to enable it to penetrate tumor cells and target intracellular target proteins.

[0124] For the above purpose, cell-penetrating anticancer peptides A to J, in which the anticancer active peptide sequence ACP (SEQ No. 14) or KLAK (SEQ No. 15) and the cell-penetrating peptide sequence iRGD (SEQ No. 16), dNP2 (SEQ No. 17), mdNP2 (SEQ No. 18), mmdNP2 (SEQ No. 19), or 9R (SEQ No. 20) are linked, were prepared by attaching acetyl groups and amide groups to the N-terminus and C-terminus, respectively, and the entire amino acid sequences were synthesized by Pepmic Co., Ltd. Ovarian cancer cell line SKOV-3 was seeded into 96-well plates at a density of 4000 cells / well with 90 μl of culture medium, and then cultured for 4 hours at 37°C under 5% CO2 conditions. 10 μl of synthetic peptides A–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, followed by incubation for 36 hours at 37°C and 5% CO2. After incubation was complete, 10 μl of CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) solution was added to the well plates and reacted for 2 hours at 37°C and 5% CO2. Upon completion of the reaction, absorbance was measured using a microplate reader.

[0125] The specific combinations of anticancer peptides and cell-permeable peptides are as shown in Table 2 below.

[0126] division Anticancer peptides Cell-permeable peptides A ACP (Sequence No. 14) iRGD (Sequence No. 16) B dNP2 (Sequence No. 17) C mdNP2 (sequence number 18) D mmdNP2 (Sequence No. 19) E 9R (Sequence No. 20) F KLAK (sequence number 15) iRGD (Sequence No. 16) G dNP2 (Sequence No. 17) H mdNP2 (sequence number 18) I mmdNP2 (Sequence No. 19) J 9R (Sequence No. 20)

[0128] Figure 10a is the result of an experiment on the cell death ability of the fusion protein against cancer cells, and Figure 10b is a graph showing the cell death ability of the fusion protein against cancer cells in numerical terms.

[0129] Looking at Figure 10 above, it can be seen that the anticancer peptide ACP has excellent apoptotic ability against cancer cells ("A") when fused with iRGD, and the anticancer peptide KLAK has excellent apoptotic ability against cancer cells ("G", "H", "I", "J") when fused with dNP2, mdNP2, mmdNP2, and 9R.

[0131] The superior anticancer effect of the ACP-iRGD combination was also confirmed through other experiments. The anticancer active peptides ACP or KLAK were combined with various types of cell-penetrating peptides and treated to SK-OV-3 cells at various concentrations, and cell viability was checked. As a result, it was found that the ACP-iRGD combination showed a significantly superior effect (Fig. 11).

[0133] Example 5: Fusion of tumor target delivery vehicle, anticancer peptide, and cell-permeable peptide (scFv-ACP-iRGD)

[0134] A fusion protein was prepared by sequentially linking an anticancer peptide and a cell-permeable peptide to the tumor target delivery vehicle of the present invention, and the binding affinity of the fusion protein to the target antigen and its ability to induce apoptosis in cancer cells were to be confirmed.

[0136] 5-1. Preparation of a fusion protein by fusing a tumor target delivery vehicle, an anticancer peptide, and a cell-permeable peptide

[0137] A fusion protein was prepared by sequentially linking an anticancer peptide and a cell-permeable peptide to a tumor target delivery vehicle (Fig. 12). In Fig. 12, the specific sequences of the components constituting each of the fusion proteins are as shown in Table 3 below.

[0138] division amino acid sequence MBP Sequence number 21 TEV protease Sequence number 22 TEV protease cleavage sequence Sequence number 23 scFv Sequence No. 24 Myc His-Tag(8xH) HHHHHHHH ACP Sequence No. 14 iRGD Sequence number 16

[0140] In Table 3 above, "scFv" is a recombinant of the variable region of the light chain (Sequence No. 39) and the variable region of the heavy chain (Sequence No. 40) of the anti-HER2 antibody (Trastuzumab) to target HER2, connected by a linker sequence (Sequence No. 41) (Sequence No. 24).

[0141] Specifically, the amino acid sequence corresponding to the [TEV cleavage sequence-scFv-ACP-iRGD] structure of the fusion protein was identified, and the nucleic acid sequence encoding that amino acid sequence was determined by optimizing it into a codon suitable for E. coli expression.

[0142] In addition, to facilitate the deletion of ACP and iRGD, adjacent XbaI and SalI sites were added to each sequence, and a stop codon was added to the end for termination. Subsequently, for cloning, SacI and PstI sites were added to the end, and the entire nucleic acid sequence was synthesized by Cosmojintech Co., Ltd.

[0144] Meanwhile, when cpACP was fused directly to an antibody fragment or fused via the Cathapsin B cleavage site, in vitro anticancer activity was not observed (Fig. 13).

[0146] Additionally, we investigated whether the anticancer activity could be enhanced by overlapping cpACP, which exhibits direct anticancer activity. As a result, although we tested the overlapping of ACP, N-term fusion of cpACP, and overlapping of cpACP, we could not confirm an increase in anticancer activity (Fig. 14).

[0148] 5-2. Expression and Purification of Fusion Protein

[0149] ACP and iRGD were deleted from the synthesized nucleic acid sequence through XbaI and SalI site deletion, and the [MBP-TEV cleavage sequence-scFv], [MBP-TEV cleavage sequence-scFv-ACP], and [MBP-TEV cleavage sequence-scFv-ACP-iRGD] recombinant expression vectors were prepared by subcloning them into the restriction enzyme sites SacI and PstI sites of pRK793 (Addgene; Plasmid #8827), an expression vector containing the MBP nucleic acid sequence.

[0150] The nucleic acid sequence of TEV protease was PCR-copied from pRK793 by adding restriction sites SacI site and XhoI site using forward primers (5'-AACGAGCTCGGGAGAAAGCTTG TTTAAGGGGCCG-3') and reverse primers (5'-GTCGAGCTCGAGGAACCATTCATGAGTTGAGTCGCTTCCTTAACTGG-3'), and this was subcloned into the SacI site of the recombinant expression vector to prepare fusion protein expression vectors with structures [MBP-TEV-TEV cleavage sequence-scFv], [MBP-TEV-TEV cleavage sequence-scFv-ACP], and [MBP-TEV-TEV cleavage sequence-scFv-ACP-iRGD].

[0151] The fusion protein expression vector prepared above was transformed into E. coli BL21(DE3) cells by applying a heat shock of 42°C, plated on LB solid medium (Ampicillin 100 µg / 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 µg / mL) for 16 hours under conditions of OD600=1.0, IPTG 0.2 mM, and 15°C, and approximately 3 g of bacterial cells were obtained from the culture medium. Subsequently, the obtained bacterial cells were suspended in PBS to lyse the cells, and the supernatant was applied to a Ni-NTA column. The column was then washed with 25 mM imidazole, and each fusion protein was eluted with 500 mM imidazole. Escherichia coli transformed with the above fusion protein expression vector expressed fusion proteins with 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 within the E. coli to purify only [scFv], [scFv-ACP], and [scFv-ACP-iRGD], respectively.

[0152] Simply put, plasmids encoding fusion proteins of the structures [MBP-TEV-TEV cleavage sequence-scFv], [MBP-TEV-TEV cleavage sequence-scFv-ACP], and

[0124] [MBP-TEV-TEV cleavage sequence-scFv-ACP-iRGD] were each expressed in E. coli and purified using Ni-NTA chromatography with (IPTG presence / absence) Ni-NTA resin, and the purified proteins were verified using SDS-PAGE (Fig. 15).

[0153] Looking at Figure 15, [scFv], [scFv-ACP], and [scFv-ACP-iRGD] cleaved intracellularly by TEV protease were present in the soluble fraction (S) and were found to be purified in one step by Ni-NTA chromatography or pull-down method.

[0154] Meanwhile, when a plasmid encoding a protein prepared by attaching only His6 to the scFv without the MBP-TEV-TEV cleavage sequence was expressed in E. coli, most of the protein was expressed in an insoluble manner and was present in the pellet (P), and only a very small amount was expressed in a soluble manner.

[0156] 5-3. Confirmation of the binding affinity of the fusion protein to the target antigen

[0157] We intended to confirm the binding affinity of the fusion proteins scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in 5-2 above for target antigens (HER2 and SKOV3).

[0158] Specifically, HER2 (Human Epidermal growth factor receptor 2) protein (Acro biosystem, HE2-H5225) was diluted to 50 ng / mL in coating buffer (200 mM Na2CO3, 200 mM NaHCo3, pH 9.6), 100 μl was dispensed into 96-well ELISA plates, and adsorbed overnight at 4°C. Afterward, unadsorbed antigens were removed by washing three times with PBS-T (PBS, 0.05% Tween20), 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. scFv, scFv-ACP, and scFv-ACP-iRGD expressed and purified in 5-2 above were each added to each well at a concentration of 0.1 pM to 400 nM at a dose of 100 μl and incubated at room temperature for 1 hour. Unbound antibodies were removed by washing three times with PBS-T (PBS, 0.05% Tween20), and ProteinL-HRP (Genscript, M00098) (in PBS) for detecting VL was added to each well at a dose of 100 μl at room temperature at a concentration of 50 ng / mL and incubated for 1 hour. Unbound ProteinL-HRP was removed by washing three times with PBS-T (PBS, 0.05% Tween20), and TMB containing 1.5% H2O2 was added to each well at a dose of 100 μl at room temperature to induce a color reaction for 30 minutes. Subsequently, 100 μl of 1M 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 deviations at each concentration were plotted using the Prism8 program. In addition, specific binding affinity (KD value) was determined using ELISA and LSF (least-squares fit) (Fig. 16, left).

[0159] In addition, ovarian cancer cell line SKOV-3 (HER2 overexpressing cell line) cultured in a culture dish at 37℃ and 5% CO2 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 5-2 were added to each well at concentrations ranging from 0.1 pM to 400 nM, respectively, and incubated at 4°C for 30 minutes. After centrifuging the cells at 4°C and 500 g for 5 minutes, they were resuspended in 1% BSA (PBS, 1% Bovine Serum Albumin) and washed twice with 1% BSA (PBS, 1% Bovine Serum Albumin).

[0160] Subsequently, ProteinL FITC (Acrobiosystems, RPL-PF141) was diluted to 5 µg / mL in 1% BSA (PBS, 1% Bovine Serum Albumin), 100 µl was added to each well, and the cells were incubated in a dark room at 4°C for 30 minutes. After washing twice with 1% BSA (PBS, 1% Bovine Serum Albumin), the cells were centrifuged at 4°C and 500 g for 5 minutes and resuspended in 100 µl of PBS. Flow cytometry data were then obtained using a FACSCalibur flow cytometer (BD, USA), and the measurements and standard deviations were plotted using Prism8 software. Additionally, specific binding affinity (KD value) was determined using flow cytometry and the least-squares fit (LSF) (Fig. 16, right).

[0161] Looking at Fig. 16, it can be seen that the fusion protein (scFv-ACP-iRGD) prepared by sequentially linking an anticancer peptide and a cell-permeable peptide to a tumor target delivery vehicle has a higher binding affinity for target antigens (HER2 and SKOV3) compared to the tumor target delivery vehicle alone (scFv) or the fusion protein (scFv-ACP) prepared by linking an anticancer peptide to the tumor target delivery vehicle.

[0163] 5-4: Confirmation of the Apoptotic Ability of Fusion Proteins Against Cancer Cells

[0164] We aimed to confirm the cancer cell killing effects of scFv, scFv-ACP, and scFv-ACP-iRGD using SKOV3 (ovarian cancer cell line) and MDA-MB-231 (breast cancer cell line).

[0165] Ovarian cancer cell line SKOV-3 (HER2 overexpressing cell line) was seeded into 96-well plates with 90 μl of culture medium at a density of 4000 cells / well and cultured for 4 hours at 37°C and 5% CO2. Subsequently, 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, followed by culture for 36 hours at 37°C and 5% CO2. 10 μl of CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) solution was added to well plates after incubation and reacted for 2 hours at 37°C under 5% CO2 conditions. After the reaction was completed, the absorbance was measured using a microplate reader.

[0166] Looking at Fig. 17, it can be seen that scFv, scFv-ACP, and scFv-ACP-iRGD of the present invention all do not exhibit apoptotic ability against cancer cells.

[0167] These results suggest that if only the tumor target delivery vehicle (scFv) and the anticancer peptide are fused without the MMP cleavage sequence of the present invention, cell death ability may be drastically reduced due to internalization and digestion in the enosome.

[0169] Example 6: Fusion of tumor target delivery vehicle, MMP cleavage sequence, anticancer peptide, and cell-penetrating peptide (scFv-MMP-ACP-iRGD)

[0170] A fusion protein was prepared by sequentially linking an MMP cleavage sequence, an anticancer peptide, and a cell-permeating peptide to the tumor target delivery vehicle of the present invention, and the apoptotic ability of the fusion protein against cancer cells was to be confirmed.

[0172] 6-1. Comparison of Apoptotic Ability of Fusion Proteins Against Cancer Cells According to Type of MMP Cleavage Sequence

[0173] A fusion protein was prepared by sequentially linking an MMP cleavage sequence (MA18, MA15), an anticancer peptide, and a cell-permeable peptide to a tumor target delivery vehicle (see 5-1 above).

[0174] The specific sequences of the components constituting each of the above fusion proteins are as shown in Table 4 below.

[0175] division Tumor target delivery system MMP cleavage sequence Anticancer peptides Cell-permeable peptides D303 Sequence No. 24 (scFv) Sequence No. 25 (MA18) Sequence No. 14 (ACP) Sequence No. 16 (iRGD) D305 Sequence No. 26 (MA15)

[0177] After expressing and purifying the fusion protein prepared above, it was verified by SDS-PAGE (see 5-2 above) (Fig. 18).

[0178] Subsequently, the apoptotic ability of fusion proteins (D303 and D305) prepared with different MMP cleavage sequences on cancer cells was tested (see 5-4 above) (Fig. 19).

[0179] Looking at Figure 19, it can be seen that both fusion proteins D303 and D305, prepared with different MMP cleavage sequences, exhibit excellent cell death ability against cancer cells (SKOV3 and MDA-MB-231).

[0182] 6-2. Comparison of Apoptotic Ability of Fusion Proteins Prepared with Different Anticancer and Cell-Permeating Peptides Against Cancer Cells

[0183] A fusion protein was prepared by sequentially linking an MMP cleavage sequence, an anticancer peptide (ACP or KLAK), and cell-permeating peptides (mmdNP2, 9R, iRGD) to a tumor target delivery vehicle (see 5-1 above).

[0184] The specific sequences of the components constituting each of the above fusion proteins are as shown in Table 5 below.

[0185] division Tumor target delivery system MMP cleavage sequence Anticancer peptides Cell-permeable peptides D301 Sequence No. 24 (scFv) Sequence No. 25 (MA18) Sequence No. 15 (KLAK) Sequence No. 19 (mmdNP2) D302 Sequence No. 15 (KLAK) Sequence No. 20 (9R) D303 Sequence No. 14 (ACP) Sequence No. 16 (iRGD) D304 - -

[0187] After expressing and purifying the fusion protein prepared above, it was verified by SDS-PAGE (see 5-2 above) (Fig. 20).

[0188] Subsequently, the apoptotic ability of fusion proteins (D301, D302, D303, and D304) prepared with different anticancer peptides (ACP or KLAK) and cell-permeable peptides (mmdNP2, 9R, iRGD) on cancer cells was tested (see 5-4 above) (Fig. 21).

[0189] Looking at Figure 21, it can be seen that fusion proteins D301 and D303, prepared with different anticancer peptides and cell-permeable peptides, exhibit excellent cell death ability against cancer cells (SKOV3 and MDA-MB-231).

[0191] The inventors named the fusion protein (scFv / antibody-MMP-ACP-iRGD) of the tumor target delivery vehicle, MMP cleavage sequence, anticancer peptide, and cell-permeable peptide prepared in Example 6 as the DTAT (Dual-Targeting Antibody-based Therapeutics) platform. This name was devised because the tumor is targeted primarily by the tumor target delivery vehicle, and the tumor is targeted secondarily by the cell-permeable anticancer peptide targeting CP2c.

[0193] Example 7: Fusion of serum albumin-binding peptide, tumor target delivery vehicle, MMP cleavage sequence, anticancer peptide, and cell-permeating peptide (saBP-scFv-MMP-ACP-iRGD)

[0194] To verify the fusion potential of saBP to increase the in vivo half-life of dual-target antibody anticancer drugs, fusion proteins were prepared by sequentially linking a serum albumin-binding peptide, a tumor target delivery vehicle, an MMP cleavage sequence, an anticancer peptide, and a cell-permeable peptide while varying the serum albumin-binding peptide.

[0196] 7-1. Selection of Serum Albumin-Binding Peptides

[0197] The specific sequence of the serum albumin-binding peptide used in the experiment of the present invention is as shown in Table 6 below.

[0198] division amino acid sequence SA20 Ac-QRLIEDICLPRWGCLWEDDF-NH2 SA21 Ac-RLIEDICLPRWGCLWEDD-NH2 SA22 Ac-RLIEDICLPRWGCLWED-NH2 SA29 Ac-RLIEDICLPRWGCLWE-NH2 SA31 Ac-RLIEDICLPRWGCLW-NH2 SA33 Ac-RLIEDICLPRWGCL-NH2 SA35 Ac-RLIEDICLPRWGC-NH2 SA23 Ac-LIEDICLPRWGCLWED-NH2 SA24 Ac-IEDICLPRWGCLWED-NH2 SA25 Ac-EDICLPRWGCLWED-NH2 SA26 Ac-DICLPRWGCLWED-NH2 SA27 Ac-ICLPRWGCLWED-NH2 SA28 Ac-CLPRWGCLWED-NH2 SA30 Ac-IEDICLPRWGCLWE-NH2

[0200] Through a study on the 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.), sequences with excellent half-maximum binding concentration (IC50) as presented in the literature were selected to identify sequences that specifically bind to albumin.

[0201] In addition, the likelihood of expression as expected hydrophilicity when applied to a dual-target anticancer drug platform was predicted and expressed as a hydrophilicity score for comparison, and candidate sequences with excellent predicted values ​​are summarized in Table 7.

[0202] division IC50(nM) hydrophilic score SA20 260 0.05 SA21 270±110 0.18 SA22 430±170 0.01 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±1,300 0.06 SA26 3,120±660 -0.17 SA27 86,700±21,800 -0.43 SA28 400,000 -0.31 SA30 1,800±590 -0.29

[0204] 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.

[0205] division Sequence number SA20 Sequence number 27 SA21 Sequence number 28 SA22 Sequence number 29 SA29 Sequence number 30 SA31 Sequence No. 31 SA23 Sequence No. 32

[0207] 7-2. Preparation of Fusion Proteins with Different Serum Albumin-Binding Peptides

[0208] A fusion protein was prepared by sequentially linking a serum albumin-binding peptide, a tumor target delivery vehicle, an MMP cleavage sequence, an anticancer peptide, and a cell-permeable peptide while varying the serum albumin-binding peptide (see 5-1 above).

[0209] The specific sequences of the components constituting each of the above fusion proteins are as shown in Table 9 below.

[0210] division serum albumin-binding peptide Tumor target delivery system MMP cleavage sequence Anticancer peptides Cell-permeable peptides D310 - Sequence No. 24 (scFv) Sequence No. 25 (MA18) Sequence No. 14 (ACP) Sequence No. 16 (iRGD) D311 Sequence No. 27 (SA20) D312 Sequence No. 28 (SA21) D313 Sequence No. 29 (SA22) D314 Sequence No. 30 (SA29) D315 Sequence No. 31 (SA31) D316 Sequence No. 32 (SA23)

[0212] After expressing and purifying the fusion protein prepared above, it was verified by SDS-PAGE (see 5-2 above) (Figs. 22 and 23).

[0213] Looking at Figures 22 and 23, it can be seen that the expression and purification rates of the fusion proteins (D310, D311, D312, D314) fused with SA20, SA21, and SA29 are superior.

[0215] 7-3. Binding affinity of saBP-conjugated fusion protein for target antigen

[0216] The binding affinity of the fusion protein formed by fusing SA20, SA21, and SA29 expressed and purified in 7-2 above to the target antigens (HER2 and SKOV3) was confirmed (see 5-3 above) (Fig. 24).

[0217] Looking at Fig. 24, it can be seen that the fusion proteins (D310, D311, D312, D314) prepared by sequentially linking a tumor target delivery vehicle, an anticancer peptide, and a cell-permeable peptide to saBP have excellent binding affinity for target antigens and serum albumin.

[0219] 7-4. Confirmation of the apoptotic ability of saBP-conjugated fusion protein against cancer cells

[0220] The apoptotic ability of the fusion protein formed by fusing SA20, SA21, and SA29 expressed and purified in 7-2 above to cancer cells was confirmed (see 5-4 above) (Fig. 25).

[0221] Looking at FIG. 25, the fusion proteins (D310, D311, D312, D314) prepared by sequentially linking a tumor target delivery vehicle, an anticancer peptide, and a cell-permeable peptide to the saBP of the present invention all exhibit excellent apoptotic ability against cancer cells. Therefore, it can be seen that the fusion of saBP does not inhibit the apoptotic ability of the fusion proteins against cancer cells.

[0223] Additionally, the anticancer activity of D311 was confirmed in various tumor cell lines, immune cell lines, and normal cell lines. As a result, it was confirmed that D311 induces cancer cell-specific apoptosis and exhibits an anticancer effect approximately 10 times superior to cisplatin. It did not show an apoptotic effect in immune cells, normal cells, or stem cells (Fig. 26).

[0225] In addition, the CRO Q-Best Bio Co., Ltd. evaluated the nonclinical efficacy of DTAT D311 and DTAT D310 (w / o saBP) relative to Herceptin in SKOV3-transplanted nude mice under the Q3Dx8 / IV scheme. The evaluation results showed that DTAT D311 demonstrated a 15.3% reduction in tumor size and a 16.9% reduction in tumor weight compared to Herceptin. Furthermore, DTAT D311, which had the saBP sequence added to increase its half-life, exhibited superior antitumor activity compared to DTAT D310 (w / o saBP) (Fig. 28).

[0227] Example 8: DTAT platform (DTAT-MSLN) containing a mesothelin-targeting antibody

[0228] DTAT-MSLN (Dual-Targeting Antibody-based Therapeutics for Mesothelin-positive cancer) was constructed using an anti-mesothelin antibody as a tumor target delivery vehicle on the DTAT platform of Example 6 (Fig. 29). As the anti-mesothelin antibodies, 1T1G5 identified in Example 2 and anetumab (developed by Bayer) were used. The sequences of 1T1G5 and anetumab are shown in Table 10 below.

[0229] division Sequence number 1T1G5 Heavy chain Sequence number 33 1T1G5 Light chain Sequence No. 34 Anetumab Heavy chain Sequence number 37 Anetumab Light chain Sequence number 38

[0231] 8-1. Comparison of Characteristics of Three Monoclonal Anti-Mesothelin Antibodies

[0232] Sequences encoding 1T1G5 and anetumab were cloned into an expression vector, expressed and isolated, and their mass and relative affinity were compared and analyzed. As a result, it was confirmed that all three antibodies were well expressed and had excellent affinity for mesothelin (Figs. 30 and 31).

[0234] 8-2. DTAT-MSLN Production

[0235] Six dual-target anticancer substances were produced by applying the DTAT-platform to three types of mesothelin antibodies. First, a light chain expression vector, a heavy chain expression vector, a light chain-MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide fusion protein (L chain-MMP-ACP-iRGD) expression vector, and a heavy chain-MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide fusion protein (H chain-MMP-ACP-iRGD) expression vector were produced for the mesothelin antibodies (Fig. 32).

[0237] Subsequently, the corresponding expression vectors were co-transformed into CHO-s cells. At this time, if 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-transformed into the cells, and if the DAR was 4:1, the heavy chain-MMP-ACP-iRGD and light chain-MMP-ACP-iRGD expression vectors were used.

[0238] 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. Additionally, DTAT-MSLN prepared according to the mesothelin antibody used is referred to as follows: DTAT-1T1G5 and DTAT-Ane.

[0239] Proteins expressed in cells were recovered and their productivity was compared. As a result, it was confirmed that DTAT2-MSLNs were stably and repeatedly expressed, and their productivity was similar to that of the monoclonal antibody. On the other hand, DTAT4-MSLNs showed an expression rate similar to that of the monoclonal antibody, but only the heavy chain cpACP was expressed in a form that was cleaved by approximately 40% (Figs. 33 and 34). It was determined that the partial cleavage of the heavy chain cpACP was caused by the CS module cleavage enzyme expressed in CHO-S cells.

[0241] 8-3. Comparison of DTAT-MSLN binding affinities

[0242] Mesothelin generates cell surface mesothelin and megakaryocyte potentiating factor (MPF) during the maturation process. Using mesothelin and MPF, the binding affinity of three types of DTAT2-MSLNs against three types of mesothelin monoantibodies was analyzed by ELISA.

[0243] Analysis results confirmed that none of the three types of DTAT2-MSLNs bind to MPF, and the binding affinity of DTATs relative to each monoantibody did not change significantly (Fig. 35). These results indicate that applying the DTAT-platform to mesothelin monoantibodies does not impede tumor targeting.

[0245] In addition, the binding affinities of three mesothelin monoantibodies and DTAT2-MSLNs to three types of mesothelin were analyzed using ELISA and FACS. The analysis results showed that although the affinity of DTAT2-MSLNs was slightly reduced compared to the monoantibodies, the absolute affinity values ​​indicated that it did not actually impede the tumor target (Figs. 36 and 37).

[0248] 8-4. Analysis of DTAT-MSLN's Pro-Anticancer Activity and Cancer Cell-Specific Cell Death

[0249] Nine cancer cell lines (Aspc-1, BxPC-3, Capan1, Capan2, MIA-Paca2, Panc1, SNU-231, SKOV3, and A431), immune system cell line (THP-1), and normal cell line (CHOs) were seeded into plates at 25% saturation. Cells were treated with DTAT2-1T1G5 and DTAT2-Ane at concentrations of 0 to 10 μM, respectively, and cultured for 24 hours. Cell viability was evaluated using the CCK-8 assay.

[0250] As a result, it was found that DTAT-MSLN induces apoptosis in most cancer cells, but there is no difference in the survival rate of immune system cell lines and normal cell lines (Figs. 38 to 40).

[0253] 8-5. Stability evaluation of DTAT-MSLN

[0254] The stability of three types of DTAT-MSLNs in buffer and serum was confirmed. Specifically, DTAT2-1T1G5 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 time had elapsed following storage, samples were taken and degraded by SDS-PAGE.

[0255] Upon verification, it was found that when stored in a buffer, DTAT2-1T1G5 showed a degradation pattern starting from day 16 at 24°C and 37°C, and DTAT2-Ane showed a degradation pattern starting from day 16 at 24°C (Fig. 41).

[0256] In addition, when stored in human serum, DTAT2-1T1G5 was stable for more than one month at 4°C and 37°C, and showed a degradation pattern starting from day 16 at 24°C. In the case of DTAT2-Ane, it was stable for more than one month at 4°C and showed a degradation pattern starting from day 8 at 24°C and day 16 at 37°C (Fig. 42).

[0257] In summary, the three types of DTAT-MSLNs are stable for more than one month when stored in a buffer under refrigeration, and are stable for more than 8 days when stored in serum at 37°C.

[0259] In addition, the stability of three types of DTAT-MSLNs relative to Kadcyla was evaluated using ELISA and CCK-8 methods. Specifically, DTAT2-1T1G5 and DTAT2-Ane were added to buffer (0.1 M Tris-glycine, pH 7.6) or human serum, respectively, and stored. After 8 and 32 days of storage, samples were retrieved to check for degradation using ELISA, or the retrieved samples were treated with cells to determine the IC50 using the CCK-8 method.

[0260] It was confirmed that DTAT2-MSLNs maintained an affinity level of ~nM, although the affinity decreased slightly as the storage period in buffer and serum increased. Additionally, it was found that the apoptotic activity of Kadcyla and DTAT2-MSLNs decreased slightly to within ~10% as the storage period increased under buffer and serum conditions, and the stability of DTAT2-MSLNs was similar to that of Kadcyla (Figs. 43 and 44).

[0262] 8-7. Non-clinical Efficacy Evaluation of Three Types of DTAT2-MSLNs

[0263] 7-week-old BALB / c nude mice (CRL form Japan) were randomly divided into 5 groups: G1-Mock (untreated, 6 mice); G2-DTAT2-An group (2.7 mg / kg, 8 mice); G3-DTAT2-1T1G5 group (2.7 mg / kg, 8 mice); and G5-Anetumab-ravtansine group (2.7 mg / kg, 4 mice). AsPC-1 cells were administered to each mouse at a rate of 1 x 10⁶ cells per head. 7 Tumor formation was induced by inoculation. When the tumor size reached 150 to 200 mm³, each test substance was administered subcutaneously once every 3 days for a total of 8 times. During administration, the tumor size was measured twice a week, and after the test was completed, the mice were sacrificed, the tumors were excised, and their weights were measured.

[0264] When the degree of tumor growth inhibition relative to G1 (Mock) was assessed 13 days after administration of the test substance, the three types of DTAT2-MSLN inhibited tumor growth by 12-13% relative to Mock and showed ~4.2% superior anticancer activity compared to Anetumab-ravtansine (Fig. 45).

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[0274] 8-9. Checking DTAT4-MSLNs characteristics

[0275] The affinity of six types of DTAT2 / 4-MSLNs relative to each monoantibody was analyzed using ELISA and FACS. The results of the affinity analysis showed that although the affinity of DTAT2 / 4-MSLNs was slightly reduced compared to the monoantibodies, in terms of absolute affinity, it was found that there was actually no impediment to the tumor target (Fig. 49 and Table 11).

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Table 11

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[0280] Example 9: DTAT platform (DTAT2 / 4-Tra) containing a HER2-targeted antibody

[0281] 9-1. DTAT-Tra Production

[0282] 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 a mesothelin-targeting antibody.

[0283] Although the problem of partial cleavage of CS in the heavy chain of DTAT4-Tra also occurred, complete DTAT4-Tra was produced in the HEK293T expressing cell line by obtaining a cell line clone in which the CS cleavage enzyme was knocked out. In addition, complete DTAT4-Tra was produced in the CHOs expressing cell line by adding glycerol during cell culture (Fig. 51).

[0284] 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 intact form was produced (Fig. 52A).

[0285] 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. The analysis results confirmed that the application of the DTAT_platform source technology (CS-ACP-CPP) hardly reduced the affinity to the HER2 protein molecule or the HER2-overexpressing cell line SKOV3 (Fig. 52B).

[0287] 9-2. Confirmation of Anticancer Activity of DTAT-Tra

[0288] 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).

[0290] In addition, the anticancer activity of DTAT-Tra produced in various tumor cell lines, immune cell lines, and normal cell lines was confirmed. As a result, it was confirmed that DTAT2 / 4-Tra induces cancer cell-specific apoptosis and exhibits an anticancer effect approximately 10 times superior to cisplatin. It did not show an apoptotic effect in immune cells, normal cells, or stem cells (Fig. 54).

[0292] DTAT2-Tra was produced by replacing each motif representing the DTAT_platform source technology (CS-ACP-CPP) with a point mutant, and the effect of each motif on anticancer activity was confirmed. As a result, it was confirmed that cytotoxicity disappeared when even one of the motifs was replaced with a point mutant (Fig. 55).

[0293] Considering the above results, the anticancer activity of DTAT-Tra is achieved through the following mechanism (Fig. 56): 1) trastuzumab binds to HER2 to target tumor cells; 2) cleavage of cpACP occurs; 3) cpACP enters the tumor cell; and 4) apoptosis occurs when cpACP targets CP2c, an oncogene. If any of these processes are not satisfied, apoptosis does not occur. Therefore, compared to existing antibody-drug conjugates (ADCs) which have toxicity issues with non-tumor cells, DTAT-Tra exhibits excellent tumor specificity and safety for normal cells.

[0295] Example 10: DTAT platform (DTAT2 / 4-Cet) containing an EGFR target antibody

[0296] 10-1. DTAT-Cet Production

[0297] DTAT2 / 4-Cet was produced in CHO cells using the same method as in Example 8-1, but using cetuximab, an antibody targeting EGFR (Epidermal Growth Factor Receptor), instead of a mesothelin-targeting antibody. A schematic diagram of the action of DTAT2 / 4-Cet is shown in Fig. 57.

[0298] However, the DTAT platform applied using the cetuximab full antibody exhibited a problem where CS was partially cleaved from the heavy chain during expression (Figs. 58a and 58b). However, in CHOs-expressing cell lines, the complete form of DTAT2 / 4-Cet was produced by adding 20% ​​glycerol during cell culture (Fig. 58c).

[0299] The production efficiency of DTAT2 / 4-Cet was verified, and it was confirmed that the addition of 20% glycerol during cell culture did not affect the production efficiency (Figs. 59a and 59b). In addition, the anticancer efficacy of DTAT2-Cet and DTAT4-Cet in A231 cells was verified, and it was confirmed that anticancer activity increased as DAR increased (Fig. 59c).

[0301] As a result of analyzing the molecular weight of the produced DTAT2-Cet using MALDI TOF, a single peak of the expected molecular weight was detected, indicating that intact DTAT2-Cet was produced. In contrast to ADCs where the DAR does not fall into an integer ratio due to heterogeneous antibody-drug binding, DTAT2-Cet showed a single peak, confirming that the DAR is constant (Fig. 60a).

[0302] The relative affinity of DTAT2-Cet for EGFR1 protein or cell line A431 overexpressing EGFR1 was analyzed using ELISA and FACS methods. The results of the affinity analysis confirmed that the change in affinity of DTAT2-Cet compared to the monoclonal antibody was minimal (Figs. 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 and EGFR1 overexpressing cell line A431.

[0304] 10-2. Confirmation of Anticancer Activity of DTAT2 / 4-Cet

[0305] The anticancer activity of the produced DTAT2 / 4-Cet was confirmed in various tumor cell lines, immune cell lines, and normal cell lines.

[0306] As a result, it was confirmed that DTAT2 / 4-Cet induces cancer cell-specific apoptosis and exhibits an anticancer effect approximately 10 times superior to cisplatin. It did not show apoptotic effects in immune cells, general cells, or stem cells (Fig. 61).

[0307] In addition, anticancer activity increased in the order of DTAT-D351 (applying the DTAT platform to Cet scFv), DTAT2-Cet, and DTAT4-Cet, and it was confirmed that anticancer activity increased as DAR increased.

[0309] DTAT2-Cet was produced by replacing each motif representing the DTAT_platform source technology (CS-ACP-CPP) with a point mutant, and the effect of each motif on anticancer activity was confirmed. As a result, it was confirmed that cytotoxicity disappeared when even one of the motifs was replaced with a point mutant (Fig. 62).

[0310] Considering the above results, the anticancer activity of DTAT-Cet is achieved through the following mechanism (Fig. 63): 1) cetuximab binds to EGFR to target tumor cells; 2) cleavage of cpACP occurs; 3) cpACP enters the tumor cell; and 4) apoptosis occurs when cpACP targets CP2c, an oncogene. If any of these processes are not satisfied, apoptosis does not occur. Therefore, compared to existing antibody-drug conjugates (ADCs) which have toxicity issues with non-tumor cells, DTAT-Tra exhibits excellent tumor specificity and safety for normal cells.

Claims

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

6. Claim 2 In claim 1, the mesothelin-specific antibody or its antigen-binding fragment 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.

7. Claim 3 In claim 1, the antigen-binding fragment is a mesothelin-specific antibody or its antigen-binding fragment 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). Claim 4 In paragraph 3, the scFv is a mesothelin-specific antibody or its antigen-binding fragment in which a light chain variable region and a heavy chain variable region are connected by a peptide linker. Claim 5 In paragraph 4, the peptide linker is a mesothelin-specific antibody or its antigen-binding fragment using the sequence of SEQ ID NO. 11 (GGGSSRSSSSGGGGSGGGG). Claim 6 A polynucleotide encoding a mesothelin-specific antibody or its antigen-binding fragment according to any one of claims 1 to 5. Claim 7 An expression vector comprising the polynucleotide of claim 6. Claim 8 A transgenic organism other than a human into which the expression vector of Paragraph 7 has been introduced. Claim 9 A pharmaceutical composition for the prevention or treatment of cancer comprising a mesothelin-specific antibody or an antigen-binding fragment thereof according to claim 1, wherein the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, peritoneal cancer, endometrial cancer, and cervical cancer. Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete

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