Dual-targeted anticancer agent and producion method therefor

Abstract

The present invention relates to a dual-targeted anticancer agent that targets both a cancer cell-specific receptor and CP2c, and a production method therefor. The dual-targeted anticancer agent exhibits excellent target specificity by binding to a cancer cell-specific target and exhibits excellent anticancer efficacy by targeting CP2c, which is an intracellular protein, and thus can be effectively used for the treatment of cancer.

Description

Dual-target anticancer drug and method of producing the same

[0001] The present invention relates to a dual-target anticancer agent targeting human epidermal growth factor receptor 2 (HER2) and CP2c, or epidermal growth factor receptor 1 (EGFR1) and CP2c, and a method for producing the same.

[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 to inhibit the growth and proliferation of cancer cells. In particular, as many tumor surface antigens (TSAs) have been identified and validated as anticancer target molecules, targeted immunotherapies utilizing monoclonal antibodies that target TSAs are becoming a major method of cancer treatment.

[0003] Meanwhile, molecular-targeted agents (MTAs) serve as a cornerstone of precision or personalized medicine for cancer treatment. Targeted therapy is currently used as a first-line treatment for various human cancers because it is expected to have fewer side effects and higher therapeutic efficacy compared to conventional chemotherapy.

[0004] Under the circumstances described above, the inventors investigated a method to enhance the anticancer effect and targeting ability of an anticancer drug, and completed the present invention by sequentially combining an anti-HER2 antibody with a linker sequence (MMP) that is specifically cleaved in the tumor microenvironment and an anticancer peptide that targets CP2c, a toxic tumor protein.

[0005] Accordingly, the object of the present invention is an antibody that specifically binds to Human Epidermal Growth Factor Receptor 2 (HER2); and

[0006] The invention provides a tumor dual-targeting fusion protein comprising one or more anticancer peptide modules connected to one end of the light chain and / or heavy chain of an antibody.

[0007]

[0008] In addition, the inventors investigated a method to enhance the anticancer effect and targeting ability of an anticancer drug, and developed a method to combine an anti-EGFR1 antibody and a cell-permeable anticancer peptide targeting CP2c, thereby completing the present invention.

[0009] Accordingly, the object of the present invention is to provide a tumor dual-targeting fusion protein comprising: an antibody that specifically binds to epidermal growth factor receptor 1 (EGFR1); and one or more anticancer peptide modules connected to one end of the light chain and / or heavy chain of said antibody.

[0010] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising a tumor dual-targeting fusion protein as an active ingredient.

[0011] [DTAT-TRA]

[0012] To achieve the above objective, one aspect of the present invention is

[0013] An antibody that specifically binds to Human Epidermal Growth Factor Receptor 2 (HER2); and

[0014] The present invention provides a tumor dual-targeting fusion protein comprising one or more anticancer peptide modules connected to one end of the light chain and / or heavy chain of the above antibody.

[0015] The above anticancer peptide module is

[0016] MMP cleavage sequence represented by the amino acid sequence of SEQ ID NO. 7;

[0017] An anticancer peptide represented by the amino acid sequence of SEQ ID NO. 8, linked to the C-terminus of the MMP cleavage sequence; and

[0018] It is characterized by the sequential connection of cell-permeating peptides represented by the amino acid sequence of SEQ ID NO. 9, which are linked to the C-terminus of the anticancer peptide.

[0019] The inventors named the tumor dual-targeting fusion protein capable of inducing specific cell death in cancer cells expressing the human epidermal growth factor receptor 2 as DTAT-Tra.

[0020] 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 whole antibodies. Additionally, the term includes chimeric antibodies, humanized antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabadies, triabadies, and tetraabadies.

[0021] According to one embodiment of the present invention, it is preferable that the antibody specifically binding to human epidermal growth factor receptor 2 is a full-length antibody. A full-length antibody refers to an antibody having the entire structure of an immunoglobulin (Ig) molecule and is composed of two heavy chains (H chain) and two light chains (L chain). In terms of function, it includes both an antigen binding site (Fab) and an efficacy site (Fc).

[0022] In addition, the full-length antibody that specifically binds to the human epidermal growth factor receptor 2 may be selected from the group consisting of trastuzumab, pertuzumab, and magetuximab, but any antibody known to bind to HER2 in the art to which the present invention belongs may be used without limitation. In one embodiment of the present invention, trastuzumab was used.

[0023] Trastuzumab (product name Herceptin) is a humanized monoclonal antibody that targets subdomain IV of HER2. It induces HER2 signaling inhibition and antibody-dependent cellular cytotoxicity (ADCC) and is used to treat early and metastatic HER2-positive breast cancer and advanced gastric cancer.

[0024] Pertuzumab is an antibody specialized in blocking the formation of HER2 homo / hetero dimers by binding to the domain II ('dimerization arm') of HER2, and exhibits a mechanism complementary to trastuzumab.

[0025] Margetuximab is an antibody that recognizes domain IV of HER2, just like trastuzumab, but has its activity enhanced by engineering modification of the Fc.

[0026] Full-length antibodies are difficult to express and have the problem of being difficult to penetrate large tumors or tissues because they are larger than antigen-binding fragments or other antibodies. However, the inventors have solved the above disadvantages by introducing multiple anticancer peptide modules to the carboxyl ends of the heavy or light chains of full-length antibodies.

[0027] Specifically, the anticancer peptide module comprises a tumor-specific cleavage linker sequence (CLS; MMP cleavage sequence) and a CP2c targeting inhibitor (CP2c targeting inhibitor; CPTin). Additionally, CPTin consists of an anticancer peptide and a cell-penetrating peptide, and the anticancer peptide can penetrate into the tumor cell through the cell-penetrating peptide.

[0028] In the present invention, the antibody and the anticancer peptide module may have a drug-to-antibody ratio (DAR) of 1:1 to 10:1, preferably 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Most preferably, the drug-to-antibody ratio (DAR) may be 2:1 to 8:1.

[0029] The above DAR refers to the ratio of a drug (anticancer peptide module) that binds to one molecule of antibody. For example, if the DAR is 2:1, it means that two anticancer peptide modules bind to one molecule of antibody, and they may bind to the light chain and / or heavy chain ends of the antibody.

[0030] Additionally, if the DAR is 8:1, the anticancer peptide module can be modified into an MMP-cleavage sequence, an anticancer peptide (COT), a linker (Sequence No. 12), an anticancer peptide (COT), and a cell-permeable peptide (iRGD) (Fig. 2a).

[0031]

[0032] Another aspect of the present invention provides a polynucleotide encoding a tumor dual-targeting fusion protein (DTAT-Tra), an expression vector comprising said polynucleotide, and a transformant into which the expression vector has been introduced.

[0033] According to one embodiment of the present invention, the polynucleotide encoding the tumor dual-targeting fusion protein may have a light chain and a heavy chain each consisting of the sequences of SEQ ID NOs 22 and 23, or a sequence containing said sequences.

[0034] Alternatively, the polynucleotide encoding the above-mentioned tumor dual-targeting fusion protein may code for the following amino acid sequence.

[0035] DTAT2H-Tra

[0036] (a) a heavy chain comprising an amino acid sequence in which the amino acid sequence of SEQ ID NO. 3 and the amino acid sequence of SEQ ID NO. 4 are supplemented with the amino acid sequence of SEQ ID NO. 7 to 9; and

[0037] (b) A light chain comprising the amino acid sequences of SEQ ID NOs 5 and 6.

[0038] DTAT2L-Tra

[0039] (a) a heavy chain comprising the amino acid sequences of SEQ ID NO. 3 and SEQ ID NO. 4; and

[0040] (b) A light chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7 to 9 are added to the amino acid sequence of SEQ ID NO. 5 and the amino acid sequence of SEQ ID NO. 6.

[0041] DTAT4-Tra

[0042] (a) a heavy chain comprising an amino acid sequence in which the amino acid sequence of SEQ ID NO. 3 and the amino acid sequence of SEQ ID NO. 4 are supplemented with the amino acid sequence of SEQ ID NO. 7 to 9; and

[0043] (b) A light chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7 to 9 are added to the amino acid sequence of SEQ ID NO. 5 and the amino acid sequence of SEQ ID NO. 6.

[0044] DTAT8-Tra

[0045] (a) a heavy chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7, 8, 12, 8 and 9 are sequentially added to the amino acid sequence of SEQ ID NO. 3 and the amino acid sequence of SEQ ID NO. 4; and

[0046] (b) A light chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7, 8, 12, 8 and 9 are sequentially added to the amino acid sequence of SEQ ID NO. 5 and the amino acid sequence of SEQ ID NO. 6.

[0047] In addition, the polynucleotide encoding the tumor dual-targeting fusion protein may be a sequence encoding the amino acid sequences of SEQ ID NOs 10 and 11.

[0048] Additionally, the expression vector may further include an HSASP (human serum albumin signal peptide) coding sequence (Sequence No. 13) at the N-terminus of the fusion protein coding sequence.

[0049] Meanwhile, a vector containing a polynucleotide encoding a tumor dual-targeting fusion protein may include a polynucleotide encoding either the heavy or light chain of an antibody, or a polynucleotide encoding both the heavy and light chains. If the polynucleotides encoding the heavy or light chains of the antibody are present 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. When the vectors are introduced into separate transformants, the expressed antibody light chains and antibody heavy chains can be recovered to produce intact antibodies.

[0050] In the present invention, an expression vector comprising a polynucleotide encoding the tumor dual-targeting fusion protein 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.

[0051] 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.

[0052] 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.

[0053] The above expression vector can produce a tumor dual-targeting fusion protein (DTAT-Tra) through the following process:

[0054] A step of transforming a host cell with an expression vector; and

[0055] A step of culturing host cells in a medium supplemented with 15 to 30% glycerol.

[0056] In general, because full-length antibodies or fusion proteins containing them are large, even when produced as recombinant proteins, modifications such as cleavage and aggregation are prone to occur during the production process.

[0057] The inventors also confirmed that partial cleavage occurs in the heavy chain (HC) of DTAT2H-Tra and DTAT4-Tra (Fig. 2B), and confirmed that adding glycerol to the culture medium resolves this cleavage problem.

[0058] According to one embodiment of the present invention, the glycerol concentration may be 15 to 30%, preferably 20% to 30%, and most preferably 25%.

[0059]

[0060] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising, as active ingredients, a tumor dual-targeting fusion protein (DTAT-Tra), a polynucleotide encoding the tumor dual-targeting fusion protein, and / or an expression vector comprising said polynucleotide, and a transformant into which the expression vector has been introduced.

[0061] In the present invention, it is preferable that the cancer be human epidermal growth factor receptor 2 (HER2) positive. HER2-positive cancers include breast cancer, colorectal cancer, endometrial cancer, ovarian cancer, lung cancer, cholangiocarcinoma, small intestine cancer, and head and neck cancer, but any cancer known to be HER2-positive in the technical field to which the present invention belongs may be applied without limitation.

[0062] In addition to containing a tumor dual-targeting fusion protein as an active ingredient, the above pharmaceutical composition may further include a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceuticals.

[0063] 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.

[0064] 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.

[0065] The composition of the present invention may be administered 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.

[0066] 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.

[0067]

[0068] Another aspect of the present invention provides a method for treating cancer comprising the step of administering a pharmaceutical composition for treating cancer, comprising the tumor dual-targeting fusion protein (DTAT-Tra) and / or the tumor dual-targeting fusion protein, to an individual requiring treatment.

[0069] The inventors confirmed that DTAT2 / 4 / 8-Tra exhibits excellent anticancer activity in various tumor cell lines and also confirmed that DTAT4-Tra has an excellent tumor growth inhibitory effect in tumor animal models. Therefore, the DTAT2 / 4 / 8-Tra of the present invention can be usefully utilized in pharmaceutical compositions for cancer treatment and for cancer treatment purposes.

[0070]

[0071] [DTAT-CET]

[0072] To achieve the above objective, one aspect of the present invention is

[0073] An antibody that specifically binds to epidermal growth factor receptor 1 (EGFR1); and

[0074] The present invention provides a tumor dual-targeting fusion protein comprising one or more anticancer peptide modules connected to one end of the light chain and / or heavy chain of the above antibody.

[0075] The above anticancer peptide module is

[0076] MMP cleavage sequence represented by the amino acid sequence of SEQ ID NO. 7;

[0077] An anticancer peptide represented by the amino acid sequence of SEQ ID NO. 8, linked to the C-terminus of the MMP cleavage sequence; and

[0078] It is characterized by the sequential connection of cell-permeating peptides represented by the amino acid sequence of SEQ ID NO. 9, which are linked to the C-terminus of the anticancer peptide.

[0079] The inventors named the tumor dual-targeting fusion protein capable of inducing cancer cell-specific apoptosis as DTAT-Cet. Specifically, DTAT-Cet comprises an antibody (cetuximab) targeting EGFR and an anticancer peptide module. The anticancer peptide module consists of a tumor-specific cleavage linker sequence (CLS; MMP cleavage sequence) and a CP2c targeting inhibitor (CP2c targeting inhibitor; CPTin), wherein CPTin consists of a cytotoxic peptide (COT) and a cell-permeable peptide (CPP).

[0080] As shown in Fig. 15, DTAT-Cet is a single protein composed of cetuximab as a carrier, an MMP11 cleavage linker sequence (CLS) which is a cancer cell-specific cleavage sequence, a CP2c oligomerization domain targeting motif (COT) which targets CP2c, a tumor protein overexpressed in cancer cells, and a cell-penetrating peptide (CPP) that transports COT into the cell. When DTAT-Cet targets the epidermal growth factor receptor (EGFR) overexpressed in cancer cells, the MMP11 CLS is cleaved, and COT penetrates into the cell via iRGD CPP to convert the CP2c complex into CP2c monomers. The converted CP2c monomers induce apoptosis in cancer cells.

[0081] 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 whole antibodies. Additionally, the term includes chimeric antibodies, humanized antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabadies, triabadies, and tetraabadies.

[0082] According to one embodiment of the present invention, it is preferable that the antibody specifically binding to human epidermal growth factor receptor 1 is a full-length antibody. A full-length antibody refers to an antibody having the entire structure of an immunoglobulin (Ig) molecule and is composed of two heavy chains (H chain) and two light chains (L chain). In terms of function, it includes both an antigen binding site (Fab) and an efficacy site (Fc).

[0083] In addition, the full-length antibody that specifically binds to the human epidermal growth factor receptor 1 may be selected from the group consisting of cetuximab, panitumumab, nimotuzumab, matuzumab, necitumumab, and zalutumumab, but any antibody known to bind to EGFR1 in the art to which the present invention belongs may be used without limitation. In one embodiment of the present invention, cetuximab was used.

[0084] Cetuximab is a chimeric IgG1 antibody characterized by strong antibody-dependent cell-mediated cytotoxicity (ADCC) activity due to the nature of IgG1. It has been approved as a treatment for metastatic colorectal cancer and head and neck cancer.

[0085] Panitumumab is a fully human IgG2 antibody characterized by low immunogenicity and very low ADCC efficacy. It has been approved as a treatment for metastatic colorectal cancer.

[0086] Nimotuzumab is a humanized IgG1 antibody characterized by moderate affinity for EGFR and low binding affinity to normal tissues. As a result, it has a relatively low incidence of skin rash and toxicity. Matuzumab is also a humanized IgG1 antibody.

[0087] Necitumumab is a humanized IgG1 antibody characterized by its ability to block EGFR while also enabling ADCC. It has been approved as a treatment for squamous cell non-small cell lung cancer.

[0088] Zalutumumab is a human IgG1 antibody and is characterized by its high affinity for EGFR.

[0089] Full-length antibodies are difficult to express and difficult to penetrate into large tumors or tissues because they are larger in size compared to antigen-binding fragments or other antibodies. However, the inventors have solved the above disadvantages by introducing multiple anticancer peptide modules to the carboxy terminals of the heavy or light chains of a full-length antibody that specifically binds to human epidermal growth factor receptor 1.

[0090] Accordingly, in the present invention, the antibody that specifically binds to human epidermal growth factor receptor 1 and the anticancer peptide module may have a drug-to-antibody ratio (DAR) of 1:1 to 10:1, preferably 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Most preferably, the drug-to-antibody ratio (DAR) may be 1:1 to 8:1.

[0091] The above DAR refers to the ratio of a drug (anticancer peptide module) that binds to one molecule of antibody. For example, if the DAR is 2:1, it means that two anticancer peptide modules bind to one molecule of antibody, and they may bind to the light chain and / or heavy chain ends of the antibody.

[0092] In addition, if DAR is 8:1, the anticancer peptide module can be modified into an MMP-cleavage sequence, an anticancer peptide (COT), a linker (Sequence No. 12), an anticancer peptide (COT), and a cell-permeable peptide (iRGD).

[0093]

[0094] Another aspect of the present invention provides a polynucleotide encoding a tumor dual-targeting fusion protein (DTAT-Cet), an expression vector comprising said polynucleotide, and a transformant into which the expression vector has been introduced.

[0095] According to one embodiment of the present invention, the polynucleotide encoding the DTAT-Cet may have a light chain and a heavy chain each composed of the sequences of SEQ ID NOs. 33 and 34, or a sequence containing said sequences.

[0096] In addition, the polynucleotide encoding the above DTAT-Cet may be a sequence encoding the following amino acid sequence.

[0097] DTAT2H-Cet

[0098] (a) a heavy chain comprising an amino acid sequence in which the amino acid sequence of SEQ ID NO. 26 and the amino acid sequence of SEQ ID NO. 4 are supplemented with the amino acid sequence of SEQ ID NO. 7 to 9; and

[0099] (b) A light chain containing the amino acid sequence of SEQ ID NO. 27.

[0100] DTAT2L-Cet

[0101] (a) a heavy chain comprising the amino acid sequences of SEQ ID NO. 26 and SEQ ID NO. 4; and

[0102] (b) A light chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7 to 9 are added to the amino acid sequence of SEQ ID NO. 27.

[0103] DTAT4-Cet

[0104] (a) a heavy chain comprising an amino acid sequence in which the amino acid sequence of SEQ ID NO. 26 and the amino acid sequence of SEQ ID NO. 4 are supplemented with the amino acid sequence of SEQ ID NO. 7 to 9; and

[0105] (b) A light chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7 to 9 are added to the amino acid sequence of SEQ ID NO. 27.

[0106] DTAT8-Cet

[0107] (a) a heavy chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7, 8, 12, 8 and 9 are sequentially added to the amino acid sequence of SEQ ID NO. 26 and the amino acid sequence of SEQ ID NO. 4; and

[0108] (b) A light chain comprising an amino acid sequence in which the amino acid sequences of SEQ ID NO. 7, 8, 12, 8 and 9 are sequentially added to the amino acid sequence of SEQ ID NO. 27.

[0109] In addition, the polynucleotide encoding the tumor dual-targeting fusion protein may be a sequence encoding the amino acid sequences of SEQ ID NOs 28 and 29.

[0110] According to one embodiment of the present invention, when a plurality of anticancer peptide modules are included in the tumor dual-targeting fusion protein, the polynucleotide encoding the tumor dual-targeting fusion protein may have the sequences of SEQ ID NOs 19 to 21 added 1 to 10 times to the sequences of SEQ ID NOs 33 and 34.

[0111] Or, if the tumor dual-targeting fusion protein contains a plurality of anticancer peptide modules, the polynucleotide encoding the tumor dual-targeting fusion protein may be a sequence encoding an amino acid sequence in which the amino acid sequences of SEQ ID NOs 7 to 9 are added 1 to 10 times to the amino acid sequences of SEQ ID NOs 28 and 29.

[0112] Additionally, the above expression vector may additionally include an HSASP (human serum albumin signal peptide) coding sequence (Sequence No. 13).

[0113] The above DTAT-Cet expression vector can produce a tumor dual-targeting fusion protein through the following process:

[0114] A step of culturing host cells into which an expression vector has been introduced in a medium to which 5 to 30% glycerol is added and the pH is 9 to 10.

[0115] In general, because full-length antibodies or fusion proteins containing them are large, even when produced as recombinant proteins, modifications such as cleavage and aggregation are prone to occur during the production process.

[0116] The inventors also confirmed that partial cleavage occurs in the heavy chain during the production process of DTAT2H-Cet and DTAT2L-Cet (Fig. 10B), and confirmed that adding glycerol to the culture medium resolves this cleavage problem.

[0117] According to one embodiment of the present invention, the glycerol concentration may be 15 to 30%, preferably 20% to 30%, and most preferably 25%.

[0118] Among the terms or elements mentioned in the above DTAT-Cet expression vector, those that are the same as those mentioned in the description of the above DTAT-Tra expression vector are understood to be the same as those mentioned in the description of the above DTAT-Tra expression vector.

[0119]

[0120] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising, as active ingredients, a tumor dual-targeting fusion protein (DTAT-Cet), a polynucleotide encoding the tumor dual-targeting fusion protein, and / or an expression vector comprising said polynucleotide, and a transformant into which the expression vector has been introduced.

[0121] In the present invention, the cancer is preferably human epidermal growth factor receptor 1 (EFGR1) positive, and specifically may be EGFR1-positive head and neck cancer, EGFR1-positive colorectal cancer, and EGFR1-positive tumor.

[0122] Among the terms or elements mentioned in the above-mentioned pharmaceutical composition containing DTAT-Cet, those that are the same as those mentioned in the description of the above-mentioned pharmaceutical composition containing DTAT-Tra are understood to be the same as those mentioned in the description of the above-mentioned pharmaceutical composition containing DTAT-Tra.

[0123]

[0124] Another aspect of the present invention provides a method for treating cancer comprising the step of administering a pharmaceutical composition for treating cancer, comprising the tumor dual-targeting fusion protein (DTAT-Cet) and / or the tumor dual-targeting fusion protein, to an individual requiring treatment.

[0125] The inventors confirmed that DTAT2 / 4 / 8-Cet exhibits excellent anticancer activity in various tumor cell lines (Fig. 13), and also confirmed that DTAT4-Cet has an excellent tumor growth inhibitory effect in an animal model with an EGFR1-positive tumor (Fig. 20). Therefore, the DTAT2 / 4 / 8-Cet of the present invention can be usefully used in pharmaceutical compositions for cancer treatment and for cancer treatment purposes.

[0126] A dual-target anticancer agent according to one example of the present invention can be effectively used in cancer treatment because it has excellent anticancer effects by targeting both epidermal growth factor receptor 1 (EGFR1) and CP2c, or human epidermal growth factor receptor 2 (HER2) and CP2c.

[0127] Figure 1 schematically shows the mechanism of action of trastuzumab-CPTin (DTAT2-Tra, DTAT4-Tra and DTAT8-Tra).

[0128] Figure 2 shows the recombinant production process of trastuzumab-CPTin:

[0129] (A) Schematic diagram of the recombinant production of trastuzumab and DTAT-Tra proteins. Tra is trastuzumab, LC is light chain, HC is heavy chain, CS is cleavage site, COT is CP2c OD (oligomerization domain)-targeting motif, LK is linkage site, L+ and H+ show CS-CPTin attached to LC and HC, respectively, and L++ and H++ show CS-COT-LK-CPTin attached to LC and HC, respectively;

[0130] (B) SDS-PAGE image of purified protein expressed in CHO-S cells. M is the molecular size marker, R is the reference protein;

[0131] (C) SDS-PAGE images of DTAT4-Tra expressed in wild-type (WT) and MMP11 knockout (KO) HEK-293T cells, and the inset (right panel) shows DNA sequencing results of KO mutant cells confirming the frameshift of the MMP11 gene;

[0132] (D) SDS-PAGE showing the effect of glycerol supplementation medium on DTAT4-Tra expression in CHO-S cells;

[0133] (E) SDS-PAGE image of purified protein expressed in CHO-S cells in glycerol-supplemented medium;

[0134] (F) Comparison of production yields in 1L of CHO-S cell culture medium; and

[0135] (G) WT and mutant DTAT2-Tra [CS m (L242D). COT m (Q250N). iRGD m (D258A)] SDS-PAGE for protein production.

[0136] Figure 3 shows the results confirming the structure and function of the synthesized trastuzumab-CPTin:

[0137] (A) Results of measuring the hydrodynamic magnitudes of DTAT2-Tra and DTAT4-Tra using dynamic light scattering (DLS);

[0138] (B) Results of measuring the molecular weights of DTAT2-Tra and DTAT4-Tra by mass spectrometry;

[0139] (C) Results confirming the HER2 targeting ability of DTAT2-Tra and DTAT4-Tra by enzyme immunoassay (ELISA) and flow cytometry;

[0140] (D) Results of analyzing the amount of CPTin cleavage in the culture medium over time by SDA-PAGE after treating normal and cancer cells with DTAT4-Tra;

[0141] (E) Results of analyzing the peptide fragment (CPTin) cleaved over time in the cancer cell culture medium of (D) above using HPLC (high-performance liquid chromatography);

[0142] (F) results of analyzing the mass of the peptide fragment (CPTin) identified in the culture medium of (D) above; and

[0143] (G) Results comparing the cytotoxicity of trastuzumab-CPTin (DTAT8-Tra, DTAT4-Tra, DTAT2-Tra, and DTAT-D311) against HER2-overexpressing SK-OV-3 cells with the cytotoxicity of competing drugs.

[0144] Figure 4 shows the results of evaluating the anticancer activity of trastuzumab-CPTin:

[0145] (A) Results of evaluating the cytotoxicity of trastuzumab-CPTin against SK-OV-3 cells by staining dead cells with propidium iodine (PI):

[0146] (B) Results of staining cells treated with trastuzumab-CPTin with PI and Annexin V and confirming by flow cytometry;

[0147] (C) Results of staining cells treated with trastuzumab-CPTin with PI and Annexin V and confirming them with a fluorescence microscope; and

[0148] (D) Results of comparative analysis of anticancer activity after treating 25 different cells with trastuzumab-CPTin.

[0149] Figure 5 shows the results of evaluating the structural and functional stability of trastuzumab-CPTin under various conditions:

[0150] (A) Results confirming the unfolding start temperature of trastuzumab-CPTin through fluorescence changes according to temperature;

[0151] (B) Results of confirming the thermal aggregation onset temperature of trastuzumab-CPTin using static light scattering (SLS);

[0152] (C) Results of confirming the size of trastuzumab-CPTin at 25℃ using DLS;

[0153] (D) Results of confirming target affinity by ELISA after storing Herceptin in PBS at 4℃;

[0154] (E) Results of confirming target affinity by ELISA after storing Herceptin in human serum at 37℃;

[0155] (F) Results of confirming target affinity by ELISA after storing DTAT4-Tra in PBS at 4℃;

[0156] (G) Results of confirming target affinity by ELISA after storing DTAT4-Tra in human serum at 37℃;

[0157] (H) Results of confirming the apoptotic activity against SK-OV-3 cells after storing Herceptin in PBS at 4℃ using the CCK-8 assay;

[0158] (I) Results of confirming the apoptotic activity against SK-OV-3 cells after storing Herceptin in human serum at 37℃ using the CCK-8 assay;

[0159] (J) Results of confirming the apoptotic activity against SK-OV-3 cells by the CCK-8 assay after storing DTAT4-Tra in PBS at 4℃;

[0160] (K) Results of confirming the apoptotic activity against SK-OV-3 cells by the CCK-8 assay after storing DTAT4-Tra in human serum at 37℃;

[0161] (L) Results of confirming the stability of DTAT4-Tra by SDS-PAGE after storing it in PBS at various temperatures (4℃, 23℃, and 37℃) and times (1 day, 2 days, 4 days, 8 days, 16 days, and 32 days); and

[0162] (M) The stability of DTAT4-Tra was confirmed by SDS-PAGE after storage in serum at various temperatures (4℃, 23℃, and 37℃) and times (1 day, 2 days, 4 days, 8 days, 16 days, and 32 days).

[0163] Figure 6 shows the results of evaluating the function of trastuzumab-CPTin in vivo:

[0164] (A) Results of checking for signs of toxicity after administering high doses of DTAT4-Tra to mice;

[0165] (B) Results of confirming in vivo behavior after administering DTAT4-Tra to mice;

[0166] (C) A protocol for evaluating in vivo antitumor efficacy after administering DTAT4-Tra to an SK-OV-3 derived xenograft mouse model; and

[0167] (D) Results of measuring tumor volume after administering DTAT4-Tra to an SK-OV-3 derived xenograft mouse model; and

[0168] (E) Results of comparing the tumor growth inhibition rate after administering DTAT4-Tra to an SK-OV-3-derived xenograft mouse model with that of competing drugs Herceptin, Kadcyla, and Enhertu;

[0169] Figure 7 shows the results of confirming activity in the presence of a mutation in DTAT2-Tra:

[0170] (A) CS, COT, and iRGD modules constituting trastuzumab-CPTin CS m (L242D), COT m (Q250N), iRGD m Structure of the trastuzumab-CPTin expression vector with the (D258A) mutation introduced;

[0171] (B) Results of confirming molecular weight by SDS-PAGE after producing DTAT2-Tra with each mutation; and

[0172] (C) Results of confirming the cytotoxicity of DTAT2-Tra with each mutation on SK-OV-3 cancer cell lines.

[0173] Figure 8 shows the design process, expression yield, and cytotoxicity of DTAT-D351:

[0174] (A) Cloning scheme of HER1_scFv and DTAT-D351. DNA was inserted into pcDNA3.4 plasmid using XbaI and NotI.

[0175] (B) SDS-PAGE analysis of HER1_scFv and DTAT-D351 produced and purified in CHO-S cells.

[0176] (C) The relative production yields of HER1_scFv and DTAT-D351 were calculated using B.

[0177] (D) Analysis of cell viability using CCK-8 assay. A431 cells were treated with HER1_scFv (blue line) or DTAT-D351 (red line) at concentrations up to 10 μM for 24 hours and compared with cisplatin (black line, up to 100 μM). Cell viability was measured using the CCK-8 assay, and data were presented as the mean ± standard deviation obtained from three independent experiments.

[0178] Figure 9 shows the results confirming the EGFR binding affinity of HER1 scFv and DTAT-D351:

[0179] (A) ELISA results confirming the EGFR binding affinity of HER1 scFv and DTAT-D351: mAb-Cet (positive control); and

[0180] (B) Flow cytometry results for A431 cell binding of HER1 scFv and DTAT-D351.

[0181] Figure 10 shows the production process of DTAT-Cet using cetuximab:

[0182] (A) DTAT-Cet expression vector; (B) results of expression confirmation; and (C) results of expressing DTAT-Cet while increasing glycerol concentration.

[0183] Figure 11 shows the results of verifying the cloning structure and characteristics of DTATs-Cet:

[0184] (A) Cloning scheme of DTATs-Cet;

[0185] (B and C) SDS-PAGE analysis of mAb-Cet and DTATs-Cet expressed in CHO-S cells cultured with 20% glycerol and purified using a Protein A column;

[0186] (D) Production yield of mAb-Cet and DTATs-Cet; and

[0187] (E) Results of CCK-8 analysis of A431 cells treated with cetuximab (mAb-Cet) and DTATs-Cet (up to 10 μM, 24 hours).

[0188] Figure 12 shows the results of verifying the characteristics of DTAT-Cet:

[0189] (A) DTAT2L-Cet mass spectrometry results;

[0190] (B) DTAT4-Cet mass spectrometry results;

[0191] (C) Results of affinity confirmation for EGFR1; and

[0192] (D) Results of affinity verification for A431 cells.

[0193] Figure 13 shows the results of confirming anticancer activity after treating cancer cell lines, immune cell lines, and normal cell lines with DTAT-D351, DTAT2-Cet, DTAT4-Cet, and DTAT8-Cet, respectively.

[0194] Figure 14 shows an expression vector scheme (A) in which mutations were induced in each motif constituting DTAT2-Cet, the results of confirming the expression of the mutant (B), and the results of confirming the anticancer activity of the mutant (C).

[0195] Figure 15 is a schematic diagram showing the principle by which DTAT-Cet exhibits anticancer activity.

[0196] Figure 16 shows the results of verifying the structural and functional stability of DTAT-Cet under various conditions:

[0197] (A) Results of confirming the unfolding start temperature of DTAT-Cet through fluorescence change according to temperature;

[0198] (B) Results of confirming the thermal aggregation start temperature of DTAT-Cet by static light scattering (SLS);

[0199] (C) Results of verifying the hydrodynamic diameter of DTAT-Cet at 25℃ using DLS;

[0200] (D) Results of confirming target affinity according to storage duration using ELISA after storing Erbitux in PBS at 4℃;

[0201] (E) Results of confirming target affinity according to storage duration using ELISA after storing Erbitux in human serum at 37℃;

[0202] (F) Results of confirming target affinity according to storage duration using ELISA after storing DTAT4-Cet in PBS at 4℃;

[0203] (G) Results of confirming target affinity according to storage duration using ELISA after storing DTAT4-Cet in human serum at 37℃;

[0204] (H) Results of confirming apoptotic activity according to storage duration after storing Erbitux in PBS at 4℃ using the CCK-8 assay;

[0205] (I) Results of confirming apoptotic activity according to storage duration after storing Erbitux in human serum at 37℃ using the CCK-8 assay;

[0206] (J) Results of confirming apoptotic activity according to storage duration after storing DTAT4-Cet in PBS at 4℃ using the CCK-8 assay;

[0207] (K) Results of confirming apoptotic activity according to storage duration after storing DTAT4-Cet in human serum at 37℃ using the CCK-8 assay;

[0208] (L) Results of confirming the stability of DTAT4-Cet by SDS-PAGE after storing it in PBS under various temperature (4℃, 24℃, and 37℃) and time (1 day, 2 days, 4 days, 8 days, 16 days, and 32 days);

[0209] (M) The stability of DTAT4-Cet was confirmed by SDS-PAGE after storage in serum at various temperatures (4℃, 24℃, and 37℃) and times (1 day, 2 days, 4 days, 8 days, 16 days, and 32 days).

[0210] Figure 17 shows the results confirming the cancer cell-specific cytotoxicity of DTAT-Cet:

[0211] (A) A431 (cancer cell); (B) THP-1 (immune cell); and (C) MEF (normal cell).

[0212] Figure 18 shows the results confirming the DTATs-Cet cell death-inducing activity:

[0213] (A) Fluorescence microscopy images taken at 0 min, 30 min, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours after treating A431 cells with cisplatin (25 μM), mAb-Cet (2.5 μM), DTAT2L-Cet (2.5 μM), and mDTAT2L-Cet (2.5 μM); and

[0214] (B) Graph showing fluorescence intensity (red %) after 24 hours of treatment (*p < 0.05, **p < 0.01).

[0215] Figure 19 shows the results of confirming the dose-dependent single-dose toxicity of DTAT4-Cet compared to Erbitux in in vivo experiments:

[0216] (A) Experimental plan for a single-dose toxicity test;

[0217] (B) Graph of weight change for each group during the experiment period;

[0218] (C) Water and feed intake of each group during the experiment period;

[0219] (D) Results of weight measurements for each organ: ovary, liver, spleen, kidney, adrenal gland, thymus, lung, heart, lymph nodes, brain, and pituitary gland;

[0220] (E) Blood test results for each group: Test items include red blood cells (10 6 Cells / μL), HGB (Hemoglobin; g / dL), HCT (Hematocrit; %), MCV (Mean Red Blood Cell Volume; fL), MCH (Mean Red Blood Cell Hemoglobin; pg), MCHC (Mean Red Blood Cell Hemoglobin Concentration; g / dL), PLT (Platelets; 10 3 Cells / μL), white blood cells (10 3 Cells / μL), NEU (neutrophils; %), LYM (lymphocytes; %), MONO (%), EOS (eosinophils; %) and BASO (basophils; %); and

[0221] (F) Results of serum chemistry tests after sacrifice for each group: Test items were ALT (ALanine Transaminase; U / L), AST (aspartate aminotransferase; U / L), ALP (alkaline phenyl phosphatase; U / L), LDH (lactate Dehydrogenase; U / L), TP (total Protein; g / dL), Alb (albumin; g / dL), A / G (albumin / globulin ratio), BUN (blood urea nitrogen; mg / dL), Crea (creatinine; mg / dL), and UA (uric acid; mg / dL)

[0222] Figure 20 shows the results confirming the in vivo efficacy of DTAT4-Cet in a HER1-positive MDA-MB-468_CDX mouse model:

[0223] (A) Experimental plan for the MDA-MB-468 xenotransplantation study: 8 animals per experimental group;

[0224] (B) Results of tumor volume (mm³) measurement during the experimental period;

[0225] (C) Results of confirming tumor growth (%) over time;

[0226] (D) Tumor weight at day 25 and representative image of the resected tumor; and

[0227] (E) Results of comparing the tumor growth inhibition rate of DTAT4-Cet with that of a competing drug.

[0228] 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.

[0229]

[0230] I. DTAT-Tra

[0231] Experimental method

[0232] 1. Reagents

[0233] pcDNA for protein expression TM 3.4 TOPO TM The plasmid vector was purchased from Invitrogen (#A14697, Carlsbad, CA, USA). Recombinant DNA containing chemically synthesized oligonucleotides was purchased from Bioneer (Daejeon, Korea), amplified by PCR, and inserted into the plasmid vector.

[0234] Transfection for DTATs-Tra expression is ExpiFectamine TMThe test was performed using the CHO Transfection Kit (Thermo Fisher, MA, USA). Horseradish peroxidase (HRP)-conjugated protein-A for ELISA was purchased from Cell Signaling Technology (#12291; Danvers, Massachusetts, USA), and HER2 ExD (extracellular domain) protein, His tag (EGR-H5222), and FITC-labeled protein-L (#RPL-PF141) for flow cytometry were purchased from ACROBiosystems (Seoul, Korea). FQI1 (SML0413) and cisplatin (P4394) were purchased from Sigma-Aldrich (St. Louis, MO).

[0235] Trastuzumab, trastuzumab-emtansine, and trastuzumab-deruxtecan were purchased as commercial products from Herceptin, Kadcyla, and Enhertu to serve as controls for functional analysis, while trastuzumab for productivity testing was produced in-house using the same method used to produce trastuzumab-CPTin. The DTAT-D311 protein was prepared as previously described (Ref. 24), and CPTin was produced from the E. coli BL21(DE3) strain. Cisplatin, MMAE, and lapatinib were purchased as commercial products to serve as cytotoxicity controls. All reagents were diluted to experimental concentrations in phosphate-buffered saline (PBS) for use in in vitro and in vivo experiments. Target proteins were analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Each sample was uniformly loaded, and the gel was stained with Coomassie Brilliant Blue G-250 (B7920, Sigma-Aldrich, St. Louis, MO).

[0236] Female BALB / c nude mice (5 weeks old) weighing 13–16 g were purchased from Orient Bio (Seongnam, Korea). Mice were housed under standard conditions (22±2℃, 55±5% humidity, 12-hour / 12-hour light-dark cycle), and food and water were provided free of charge. All experiments were approved and performed in accordance with the guidelines of the Konkuk University Animal Ethics Committee (#KU23263).

[0237]

[0238] 2. Cell line

[0239] AsPC1, BxPC3, Capan1, Capan2, MIA PaCa2, Panc1, and SNU213 (pancreatic cancer), SK-OV-3 (ovarian cancer), MCF7, MDA-MB-231, SKBR3 (breast cancer), A431 and A2058 (skin cancer), SNU-1041 (head and neck cancer), HT29, HCT-116, and SW480 (colorectal cancer), A-549, H441, and H460 (lung cancer), THP-1, and Jurkat (blood cancer) cell lines were purchased from the Korean Cell Line Bank (KCLB, Seoul, South Korea). Cultures were performed in high-glucose Dulbecco's modified Eagle medium or Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 μg / mL). TM ...was performed at 5% CO2. Expi CHO-S cells were purchased from Thermo Fisher (#A29127) and cultured in ExpiCHO expression medium (Thermo Fisher, #A2910001) in an 8% CO2 incubator maintained at 37°C and 110 rpm humidity. One day before transfection, cells were cultured at 3–4 x 10°C. 6 They were dispensed at a density of cells / mL. After 24 hours, the cell stock was 7–10×10 6 A density of cells / mL was reached, and then new medium was added for 6×10 6The cells were diluted to cells / mL. A mutant HEK-293T cell line deficient in the MMP11 gene was constructed by inducing a frameshift of the MMP11 gene. To this end, the sgRNA sequence of the lentiCRISPR v2-dCas9 (Addgene plasmid #112233) vector was designed to target the second exon of the MMP11 gene. Subsequently, mutant clones were selected from cells transfected with the vector construct via colony PCR for the MMP11 gene and verified by DNA sequencing. Mouse embryonic fibroblasts (MEF) were isolated from pregnant CD-1 mice (Orient Bio, Seongnam, Korea) and cultured in high-glucose DMEM supplemented with 10% FBS at 37°C and 5% CO2.

[0240]

[0241] 3. Design and Preparation of Expression Vectors for DTAT-D311 and DTATs-Tra

[0242] The basic anti-Her2 scFv protein was expressed using the recombinant plasmid v24, constructed based on the pRK793 (Addgene #8827) vector plasmid (Ref. 26). Different constructs were created by modifying the v24 plasmid by inserting oligonucleotides necessary to encode additional sequences between the appropriate restriction enzyme sites of the plasmid. All recombinant DNA constructs were verified using DNA sequencing. Expression and purification of the recombinant protein were performed according to previously established protocols (Ref. 26).

[0243] Briefly, E. coli BL21(DE3) cells transformed with a recombinant plasmid were cultured in LB medium at 37°C, and protein expression was induced using 0.5 mM isopropyl β-D-1-thiogalactopyranoside. Proteins of interest were purified from the supernatant of cell lysates using sequential nickel affinity chromatography and size exclusion chromatography. The molecular weight of the purified proteins was determined by MALDI-TOF mass spectrometry as previously described (References 26-28).

[0244]

[0245] To develop the DTATs-Tra plasmid, a chemically synthesized and codon-optimized recombinant HSASP (human serum albumin signal peptide) sequence (SEQ No. 1) and XbaI and NotI restriction sites for subsequent insertion into pcDNA3.4 via XbaI and AgeI restriction sites were used. TM TOPO TM It was inserted into a vector. Subsequently, chemically synthesized and codon-optimized sequences were inserted through the XbaI and NotI restriction enzyme sites. The sequences used are as follows, and "CPTin (CP2c targeting inhibitor) consists of a CP2c oligomerization domain targeting motif (COT) and a cell-permeating peptide (iRGD) that target CP2c.

[0246] DTAT-Tra HC: [Trastuzumab heavy chain]-[MMP11 CS]-[CPTin]

[0247] DTAT-Tra LC: [Trastuzumab light chain]-[MMP11 CS]-[CPTin]

[0248] In addition, a trastuzumab plasmid was constructed by inserting the sequence from which [MMP11 CS]-[CPTin] was removed. Also,

[0249] In addition, for the production of DTAT8-Tra, H++ and L++ plasmids were constructed by inserting the [COT]-[LK(linker)] sequence between [MMP11 CS] and [CPTin] ("H++" and "L++" in Fig. 2a).

[0250] To induce point mutations in the DTAT-Tra light chain, site-specific mutagenesis using synthetic primers was performed to construct the DTAT-Tra LC mutant MMP11 CS and DTAT-Tra LC mutant iRGD plasmids. Additionally, for the DTAT-Tra LC mutant COT, a chemically synthesized DNA fragment containing the desired mutation was inserted into the DTAT-Tra light chain plasmid using the BamHI and NotI restriction enzyme sites. All recombinant DNA constructs were verified through DNA sequencing.

[0251]

[0252] All antibody proteins were expressed by transfecting CHO-S cells and MMP11 KO CHO-S cells, and transfection was performed using ExpiFectamine TM It was performed according to the manufacturer's protocol for the CHO Transfection Kit (Thermo Fisher). Briefly, 6X10 6 CHO-S cells were transfected with plasmids encoding recombinant proteins. For the expression of all antibodies, light-chain and heavy-chain plasmids were transfected in a 1:1 ratio. For transfection, the plasmid DNA and transfection reagents were each diluted in OptiPRO SFM (Thermo Fisher), immediately mixed, and incubated for 5 minutes. The resulting transfection mixture was applied directly to CHO-S cells. After 18–20 hours, an enhancer, a feed solution, and 20% glycerol were added to the culture medium to improve protein expression and stability.

[0253] After 5 days, the cells were harvested, and the culture supernatant was collected for protein purification. The recombinant protein was HiTrap TM MabSelect TM Purification was performed using FPLC affinity chromatography with a SuRe column (Cytiva, #11003494). Proteins captured on the column were eluted with 1 M glycine-HCl (pH 2.3) and immediately neutralized with 1 M Tris-HCl buffer (pH 8). The concentration of the purified protein was precisely quantified by spectrophotometry at 280 nm using molar absorbance values ​​calculated from the amino acid sequence using the ProtParam tool (https: / web.expasy.org / protparam / ).

[0254]

[0255] Information on each antibody and sequence used in this invention is summarized in the table below.

[0256] 서열서열번호human serum albumin signal peptide(아미노산)MKWVTFISLLFLFSSAYS1HER2 scFv(아미노산)SQRLIEDICLPRWGCLWEDDFGGSRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAS2Trastuzumab VH(아미노산)LEEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS3Heavy chain Fc region(아미노산)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGS4Trastuzumab VL(아미노산)LEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK5TrastuzumabCL(아미노산)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGS6MMP11 cleavage site(아미노산)GGYAELRMGG7COT(아미노산)NYPQRP8iRGD(아미노산)CRGDKGPDC9Trastuzumab H-MMP11-COT-iRGD(아미노산)LEEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGGYAELRMGGNYPQRPGGCRGDKGPDC10Trastuzumab L-MMP11-COT-iRGD(아미노산)LEDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGGYAELRMGGNYPQRPGGCRGDKGPDC11DTAT8-TraLinker(Amino Acid)GGGSGGGGSGGGGSGGGS12

[0257]

[0258]

[0259]

[0260] 4. In vitro cytotoxicity analysis

[0261] For the cytotoxicity test, cells were plated in 90 μL of medium in 96-well plates at a cell density of approximately 5,000 cells per well. The plates were incubated for 24 hours at 37°C in a 5% CO2 humidified environment. After incubation, 10 μL of test samples, prepared by dissolving the recombinant protein and various drug formulations in PBS, were added to each well. Each compound was prepared at its maximum concentration, and then the IC50 50 Serial dilution was performed to generate a concentration-response curve for measurement. After culturing the cells for an additional 24 hours, 10 μL of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader (BioTek, Vermont, USA) to evaluate cell viability.

[0262]

[0263] 5. ELISA

[0264] For the immunoassay of HER2 scFv, trastuzumab, and DTATs-Tra, 0.5 μg / mL of HER2 Exd (ACROBiosystems) was coated onto 96-well immunoassay plates and incubated overnight at 4°C. The HER2 Exd was diluted in 100 mM bicarbonate / carbonate coating buffer (pH 9.6). To block non-specific binding, 200 μL of 1% BSA dissolved in PBS was added to each well and incubated at 25°C for 1 hour. After blocking binding, protein samples were added to the wells and incubated at 25°C for 1 hour. Next, 100 μL of HRP-Protein-L (Cell Signaling Technology) diluted 1:10,000 was added to each well and incubated for another hour at 25°C. After incubation, 100 μL of 3,3',5,5'-tetramethylbenzidine substrate solution (Thermo Scientific) was added, and the reaction was carried out at 25°C for 20 minutes. To stop the reaction, 100 μL of 1N HCl was added to each well. Absorbance was measured at 450 nm using a microplate reader (BioTek). To ensure accuracy, the immunoplate was washed three times with PBS-T (pH 7.4) throughout the entire process, including the coating, blocking, binding, detection, and reaction steps.

[0265]

[0266] 6. Flow cytometry

[0267] The antigen binding ability of protein samples on the cell surface was evaluated using flow cytometry. For the analysis, SK-OV-3 cells (approximately 10 6Cells were cultured with various concentrations of each protein sample (0.1 pM–400 nM) at 4°C for 30 minutes. After culture, cells were washed twice with flow cytometry staining buffer (1% BSA / PBS) to remove unbound proteins. To detect DTATs-Tra and trastuzumab binding, cells were cultured for an additional 90 minutes with FITC-Protein-L 5 μg / mL. After washing twice more with staining buffer, flow cytometry was performed using a Novocyte instrument (Agilent, CA, USA) for quantification.

[0268]

[0269] 7. Mass Spectrometry

[0270] The precise molecular weight of the purified protein was measured using a MALDI-TOF mass spectrometer (Ultraflextreme, Bruker Daltonics, Bremen, Germany) operating in linear mode with a 337 nm smart beam laser. Prior to analysis, the protein sample was sterilized in 70% (v / v) acetonitrile / H₂O₃ containing 0.1% trifluoroacetic acid. 2 It was prepared by mixing the solution with a sinapinic acid matrix.

[0271]

[0272] 8. Analysis of Apoptosis Types

[0273] SK-OV-3 cell death over time was monitored using propidium iodine (PI) staining. For this purpose, cells were seeded into 96-well plates (5,000 cells in 80 μL of medium per well) and incubated at 37°C for 24 hours. Then, 10 μL of PI (10 μg / mL) and 10 μL of drug solution (25 μM protein or 250 μM chemical) were added to each well, followed by an additional 24 hours of incubation. Images were captured at specified time points using a fluorescence microscope.

[0274] To distinguish the types of cell death, SK-OV-3 cells seeded in 12-well plates (50,000 cells in 900 μL of medium per well) were cultured for 24 hours and then treated with drug samples at a final concentration of 2.5 μM (protein drug) or 25 μM (chemical drug).

[0275] After 6 hours of culture, cells were analyzed using FACS and fluorescence microscopy. Before flow cytometry analysis using FACS, harvested cells were resuspended in a buffer containing annexin-V labeled with PI and FITC using the Alexa Fluor 488 Annexin V / Dead Cell Apoptosis Kit (ThermoFisher Scientific) and cultured at room temperature (approx. 20°C) for 15 minutes.

[0276]

[0277] 9. Pharmacokinetics Analysis

[0278] 10 6-week-old BALB / c nude female mice 7 Canine SK-OV-3 cells were subcutaneously injected, and xenograft mice were established after 3 weeks. Pharmacokinetics (PK) were evaluated using single-photon emission computed tomography / computed tomography (SPECT / CT) imaging and biodistribution studies. Briefly, xenograft mice for Nano-Scan SPECT / CT (Mediso, Budapest, Hungary) scanning were divided into three groups (n = 3 per group) and 125 513.10 ± 20.22 μCi / 200 μL of the drug labeled with I was intravenously injected. Drug uptake in the tumor was quantified using the standardized uptake value (SUV) calculated from the voxel intensity.

[0279]

[0280] For the biodistribution study, xenograft mice were divided into groups (n = 5 per group), and for each mouse 12510.30 ± 0.23 μCi / 200 μL of labeled drug was intravenously injected. After 0.5, 2, 6, 24, 48, 72, 120, and 168 hours, mice were euthanized to collect blood samples, and various organs and tumors were isolated. Then, the radioactivity of the blood, organ, and tumor samples was measured to calculate the percentage of the injection volume per 1 g of blood / organ / tumor (%ID / g). The area under the curve (AUC) values ​​in the %ID / g profile over time were calculated using the trapezoidal method with Phoenix WinNonlin 8.2 software (Certara).

[0281]

[0282] 10. Stability Evaluation

[0283] To investigate the stability of trastuzumab and DTAT4-Tra, the proteins were prepared at planned assay concentrations in buffer (20% glycerol in PBS) or human serum and dispensed into separate tubes for incubation at 4°C, 23°C (room temperature), or 37°C. For SDS-PAGE analysis, human serum was stripped with IgG using a HiTrap Protein A HP column (Cytiva Korea). At specified time points (days 0, 1, 2, 4, 8, and 32), each sample was flash-frozen in liquid nitrogen (LN₂) and stored at -80°C. Subsequently, SDS-PAGE, ELISA, and CCK-8 assays were performed according to the previously described protocol, maintaining a maximum CCK-8 treatment concentration of 10 μM.

[0284]

[0285] 11. Single-dose toxicity test

[0286] Acute toxicity studies were conducted in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines for chemical testing. Female mice were weighed and randomly divided into four groups: 6 mice per group for the experimental group and 4 mice per group for the vehicle group. After fasting overnight, each group was administered a single intravenous injection of the drug at doses of 0, 100, or 300 mg / kg (mpk). Signs of toxicity were monitored for two days. Water and feed were provided on a free basis, and the daily body weight and feed intake of each mouse were recorded. After the two-day observation period, the mice were sacrificed, their organs were excised and weighed, and blood samples were collected for complete blood count (CBC) and differential analysis.

[0287]

[0288] 12. Research on In Vivo Xenograft Mouse Models

[0289] The xenograft mouse model is 1.0×10 female BALB / c nude mice 7 SK-OV-3 cells were administered via subcutaneous injection. When the average tumor volume reached approximately 150 mm³, mice were randomly assigned to each group. Mice were intravenously administered a designated dose of the drug every 3 days (Q3D) for a total of 8 treatments. Tumor size and body weight were recorded every 3 days. Six days after the last (8th) treatment, mice were euthanized to evaluate the residual drug effect, and tumors were harvested and weighed.

[0290]

[0291] 13. Statistical Processing

[0292] Statistical analysis was performed using GraphPad Prism version 8.0 software (www.graphpad.com, USA) by applying appropriate statistical methods. Data were presented as mean ± standard deviation (SD) for in vitro experiments and mean ± standard error (SEM) for in vivo experiments. The concentration required for 50% inhibition (IC10) 50 ) and dissociation constant (K d ) was determined using the nonlinear least squares method. Statistical significance was evaluated using p-values. Not significant: p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0293]

[0294]

[0295] Experimental results

[0296] 1. Characteristics of Trastuzumab-CPTin and Recombinant Production

[0297] Figure 1 schematically shows the production and molecular action of trastuzumab-CPTin.

[0298] The final product, DTAT4-Tra, has four CPTin modules, which are linked to the C-terminus of the heavy and light chains of a trastuzumab antibody via a peptide linker having an enzyme cleavage site (CS). Thus, the entire DTAT4-Tra molecule can be produced as a single recombinant protein in mammalian cells such as CHO-S.

[0299] Upon in vivo administration, the mAb transporter compartment (trastuzumab) binds to tumor cells by targeting HER2, and subsequently, the CS linker compartment is cleaved by extracellular proteases particularly abundant in tumor cells, releasing the CPTin payload to the cell surface. Next, the iRGD module of the freed CPTin is absorbed into the cell via integrin and Neuropilin-1 (NRP1)-mediated endocytosis (References 14 and 15). After internalization, the COT motif of CPTin (CP2c OD (oligomerization domain)-targeting motif) targets CP2c, which is overexpressed in tumor cells. Finally, the dissociation of monomeric CP2c by the degradation of the CP2c complex (allotetramer C4 and heterotetramer C2B2P2) induces tumor cell death.

[0300]

[0301] Considering this mechanism of action, the inventors developed four types of trastuzumab-CPTin (Fig. 2A):

[0302] DTAT2L-Tra with CPTin modules extended in each light chain (LC);

[0303] DTAT2H-Tra with CPTin modules extended to each heavy chain (HC);

[0304] DTAT4-Tra with CPTin modules extended to the light and heavy chains of trastuzumab, respectively; and

[0305] DTAT8-Tra, in which COT-LK-CPTin modules are extended to the light and heavy chains of trastuzumab, respectively

[0306]

[0307] In previous studies on suitable CS linkers, the matrix metalloprotease-11 (MMP11) recognition sequence was identified as the most efficient linker peptide, so the MMP11 recognition sequence was used as a linker, as was used in the construction of DTAT-D311.

[0308] The individual polypeptide chains also shared an N-terminal fusion of human serum albumin signaling peptide (HSA-SP), which guided the extracellular secretion of the expressed protein and was subsequently removed. All of these trastuzumab-CPTin proteins were overexpressed in CHO-S cells, recovered from the culture medium, and simply purified by protein-L affinity chromatography.

[0309] Upon verification after purification, DTAT2H-Tra and DTAT4-Tra underwent partial cleavage of the HC, whereas DTAT2L-Tra remained intact (Fig. 2B). Based on the apparent size of the cleaved HC consistent with trastuzumab HC, it was hypothesized that this unexpected cleavage occurred via MMP11 cleavage of the CS linker, potentially leading to a loss of CPTin during protein expression. As a preliminary test to confirm this hypothesis, the expression of DTAT4-Tra was compared in wild-type and MMP11-knockout HEK-293T cells (Fig. 2C). Since the previously observed cleavage was prevented in selected mutant strains, it was thought that uniform production of DTAT4-Tra would be possible using host cells with the MMP11 gene deleted (References 26 and 27). However, unfortunately, the attempt to establish an MMP11 knockout clone in the CHO-S cell line, the most widely used production cell line for the mass production of mAbs including trastuzumab, was not entirely successful.

[0310] Meanwhile, the fact that the LC of DTAT2L-Tra and DTAT4-Tra remains unchanged suggests that achieving resistance to cleavage through structural stabilization may be an alternative. Therefore, to structurally stabilize the proteins, we identified suitable additives for the culture medium and confirmed that adding more than 20% glycerol was effective in completely preventing HC cleavage (Fig. 2D). Accordingly, we established production processes for all trastuzumab-CPTin in CHO-S cells and compared production yields (Fig. 2F). In particular, compared to trastuzumab, productivity of DTAT8-Tra increased by approximately 31% and DTAT4-Tra by approximately 18%, while DTAT2L-Tra and DTAT2H-Tra increased by approximately 16% and 14%, respectively. Additionally, CS (CS) used as a functional negative control m ), COT(COT m ), iRGD(iRGD m Three mutant proteins of DTAT2L-Tra with dysfunction point mutations were also well generated under established conditions (Fig. 2G).

[0311]

[0312] 2. Confirmation of the structure and function of trastuzumab-CPTin

[0313] Among the three trastuzumab-CPTin recombinant proteins, DTAT2L-Tra (hereinafter DTAT2-Tra) and DTAT4-Tra were selected as subjects for follow-up studies. The hydrodynamic sizes of DTAT2-Tra and DTAT4-Tra, measured by dynamic light scattering (DLS), were approximately 9.8 nm and 12.3 nm, respectively, which were similar to the trastuzumab product Herceptin (approx. 10.6 nm diameter) (Fig. 3A). In addition, the DLS data showed single dispersion for each protein, confirming that the purified recombinant proteins were homogeneous without aggregates.

[0314] Subsequently, mass spectrometry was used to confirm whether the protein was damaged (i.e., the maintenance of the designated linker-CPTin extension) (Fig. 3B). The estimated molecular weights of the fully integrated DTAT4-Tra (159.4 kDa) and DTAT2-Tra (152.0 kDa) were identical to the theoretical masses calculated from the entire sequence with the N-terminal signal peptide removed. The mass of the linker-CPTin peptide was also present in all other low-mass peaks that appeared due to the potential for dissociation of the polypeptide chain during measurement.

[0315]

[0316] To confirm the function, enzyme immunoassay (ELISA) and flow cytometry confirmed that the HER2 targeting ability of the trastuzumab compartment was robustly maintained in both DTAT2-Tra and DTAT4-Tra (Fig. 3C).

[0317] Next, DTAT4-Tra was treated to normal and cancer cell culture media, and the amount of CPTin cleavage was investigated over time. In contrast to robust stability in normal mouse embryonic fibroblast (MEF) cell culture media, distinct cleavage of DTAT4-Tra was observed in both HC and LC in cancer cell SK-OV-3 cell culture media (Fig. 3D).

[0318] Subsequent HPLC analysis of the reaction medium revealed the gradual appearance of peptide fragments (Fig. 3E), and mass spectrometry confirmed that they matched the C-terminal sequence (Fig. 3F). Furthermore, when a dysfunctional mutation was introduced into the CS, such cleavage did not occur. Therefore, these results clearly demonstrate that the CPTin payload was released from SK-OV-3 cells through designated CS cleavage by MMP11 secreted extracellularly.

[0319] Finally, the cytotoxicity of trastuzumab-CPTin against HER2-overexpressing SK-OV-3 cells was evaluated by comparing it to the cytotoxicity of six competing drugs via cell viability analysis (Fig. 3G). The competing drugs used were: 1) Herceptin (trastuzumab) as an anti-HER2 mAb, 2) Kadcyla (trastuzumab-emtansine) as an anti-HER2 ADC, 3) DTAT-D311 as an scFv-based anti-HER2 DTAT (Ref. 24), 4) lapatinib as a breast cancer anticancer agent, 5) cisplatin as a conventional anticancer agent, and 6) monomethylauristatin E (MMAE) as an antimitotic chemical used as a payload for various ADCs.

[0320] As with previous observations, Herceptin showed minimal cytotoxicity in this cell-level analysis, suggesting that HER2 neutralization alone does not exert anticancer activity.

[0321] On the other hand, DTAT2-Tra is an IC 50 The value was 0.88 μM, indicating potent cytotoxicity, which was lower (i.e., more potent) than cytotoxic chemicals (MMAE, cisplatin, lapatinib) and DTAT-D311. Since the anticancer cytotoxicity of DTAT2L-Tra disappears upon introducing point mutations into CS, COT, or iRGD (Fig. 7), it was supported that this cytotoxicity is mediated by CPTin release via CS cleavage, cell infiltration via iRGD, and CP2c inhibition via COT.

[0322] Also, IC 50 According to the values, DTAT4-Tra (IC50 0.17 μM) is 5.2 times more potent than DTAT2-Tra, and DTAT8-Tra (IC50 0.08 μM) is 2.1 times more potent than DTAT4-Tra. These results suggest that increasing the number of CPTin payloads (8 for DTAT8-Tra, 4 for DTAT4-Tra, and 2 for DTAT2-Tra) may increase cytotoxicity.

[0323] DTAT2-Tra's IC 50 Although it was higher than that of Kadcyla (0.19 μM) (i.e., lower efficacy), DTAT4-Tra was equivalent to or superior to Kadcyla. However, the IC50 80 When confirming this, both DTAT2-Tra and DTAT4-Tra were effective in achieving complete apoptosis, which Kadcyla failed to achieve. Therefore, trastuzumab-CPTin, particularly DTAT4-Tra, is evaluated to have superior anticancer activity compared to Kadcyla in terms of efficacy and effectiveness.

[0324]

[0325] 3. Additional Confirmation of Trastuzumab-CPTin Anticancer Activity

[0326] The cytotoxicity of trastuzumab-CPTin on SK-OV-3 cells was further evaluated by staining dead cells with propidium iodine (PI) over time. The apoptotic efficacy of DTAT-D311, evaluated using the same method, was found to be higher than that of the previously tested chemotherapy agent cisplatin and the CP2C inhibitor FQI1. Based on PI fluorescence intensity, the trastuzumab-CPTin of the present invention was found to be superior to the existing DTAT-D311 (Fig. 4A).

[0327] The superior performance of DTAT4-Tra compared to DTAT2-Tra suggests that efficacy increases with higher CPTin payloads. COT of DTAT2-Tra mThe mutations did not induce significant apoptosis, which reaffirmed that apoptosis induced by the CPTin payload is mediated by the action of COTs targeting CP2c. Additionally, as previously observed in DTAT-D311, cells treated with DTAT4-Tra and DTAT2-Tra exhibited significant PI fluorescence within the first 30 minutes, in contrast to the apoptosis previously induced after 12 hours of treatment with cisplatin. Therefore, the cytotoxic action of trastuzumab-CPTin may be characterized by the rapid onset of cancer cell death.

[0328]

[0329] The type of cell death induced by trastuzumab-CPTin was identified as follows. Specifically, cells treated with trastuzumab-CPTin were stained with Annexin V labeled with PI and fluorescein-5-isothiocyanate (FITC) and analyzed by flow cytometry (Fig. 4B) and fluorescence microscopy (Fig. 4C).

[0330] Both datasets showed that trastuzumab-CPTin primarily induces apoptosis, which is consistent with reports that functional inhibition of CP2c induces apoptosis in cancer cells (References 9 and 13). Based on the FITC to PI fluorescence ratio, the proportion of cells killed via apoptosis was approximately 90% for DTAT4-Tra and 80% for DTAT2-Tra, which was significantly higher than previously estimated values ​​for DTAT-D311 (approx. 66%) and cisplatin (approx. 31%) (Fig. 4C).

[0331]

[0332] Finally, to determine whether the cancer cell selectivity and pan-anticancer activity of CPTin in trastuzumab-CPTin are preserved, cell viability analysis was performed on 25 different cell types, similar to previous tests for DTAT-D311, cisplatin, and FQI1 (Fig. 4D). As with the previous DTAT-D311, trastuzumab-CPTin exhibited cytotoxicity in all 20 cell lines representing 7 types of cancer, but did not exhibit significant cytotoxicity in any of the 5 non-cancerous cell lines, including MEF primary cells and 2 immune cell lines.

[0333] In contrast, IC 50 DTAT4-Tra with values ​​of 0.17–2.73 μM and an average of 1.08 μM was compared to DTAT2-Tra (IC2) in all tested cancer cell lines. 50 It showed potent anticancer activity compared to (0.51–4.89 μM, average 1.88 μM). Average IC50 of DTAT4-Tra 50 (1.08 μM) was 3 times, 23 times, and 38 times lower than the previously determined values ​​for DTAT-D311 (3.29 μM), cisplatin (25.4 μM), and FQI1 (42.1 μM), respectively, which demonstrates the superiority of DTAT4-Tra.

[0334]

[0335] 4. Confirmation of the structural and functional stability of trastuzumab-CPTin

[0336] Thermal denaturation was analyzed to compare the structural stability of trastuzumab-CPTin and trastuzumab. All proteins tested showed similar unfolding onset temperatures (T) during thermal unfolding monitored by fluorescence spectroscopy. O , approximately 62°C) and the midpoint between the two melting points (T m 1 and T mIt was characterized by a two-stage transition with values ​​of approximately 71°C and 81°C. Thermal coagulation monitored by static light scattering (SLS) also showed similar intermediate coagulation temperatures (T a It showed (approx. 78°C) (Fig. 5B). These results demonstrate that the high thermal stability of trastuzumab remains robust even when extended to trastuzumab-CPTin.

[0337] In addition, considering the in vivo usability of trastuzumab-CPTin, the stability of DTAT4-Tra was verified by storing or culturing for 32 days at various temperatures, including serum conditions. As a result, except for the room temperature storage condition on day 32 of SDS-PAGE, both the heavy and light chains of DTAT4-Tra remained intact even in serum at 37°C (Fig. 5M). This suggests that there is no possibility of trastuzumab-CPTin losing the CPTin payload due to enzymatic degradation in the blood before reaching cancer cells in vivo. Finally, high functional stability of DTAT4-Tra was also secured, as HER2 binding affinity and cytotoxicity were maintained during 32 days of culture in serum at 37°C (Figs. 5G, 5K).

[0338] Prior to in vivo efficacy testing, the in vivo safety of the final product, DTAT4-Tra, was established by the fact that no significant signs of toxicity were observed in mice even at very high doses (300 mg / kg) (Fig. 6A). In addition, since the pharmacokinetic profiles measured using single-photon emission computed tomography / computed tomography (SPECT / CT) imaging were in good agreement, the in vivo behavior of DTAT4-Tra can be expected to be similar to that of Herceptin (Fig. 6B).

[0339]

[0340] Finally, the in vivo antitumor efficacy of DTAT4-Tra was evaluated by administering it a total of five times at 3-day intervals to an SK-OV-3 cell-derived xenograft mouse model, and the results were compared with Herceptin, Kadcyla, and Enhertu (Figs. 6C-6E). Kadcyla and Enhertu were limited to clinically applicable doses (4 mg / kg or 41.23 nmol / kg) considering their potential toxicity, while Herceptin and DTAT4-Tra were used at doses four times higher (164.92 nmol / kg) to reflect their relatively superior safety profiles.

[0341] Except for one mouse that dropped out due to an accident, all experimental mice showed no statistically significant change in body weight over 18 days, suggesting that there was no significant toxicity with either drug.

[0342] As a control group, the tumor volume and tumor growth inhibition rate (TGI) at day 15 for the Herceptin administration group was 40.76% compared to the untreated group (Fig. 6E). The TGIs for the Kadcyla and Enhertu administration groups were 43.10% and 65.28%, respectively, showing inhibition effects 5.75% and 60.18% higher than Herceptin, and notably, Enhertu induced tumor regression after the 6th administration (day 15).

[0343] The DTAT4-Tra administration group showed TGI of 76.75% and 91.33% at the medium dose (G6) and high dose (G7), respectively, demonstrating a significantly higher antitumor effect than Kadcyla and Enhertu. In addition, no side effects were observed despite the increase in dose, and DTAT4-Tra induced tumor regression immediately after administration. These results demonstrate that DTAT4-Tra showed improved efficacy in vivo compared to Herceptin by up to 124.09%, compared to Kadcyla by up to 111.92%, and compared to Enhertu by up to 39.90%, proving its potential as a next-generation treatment with the potential to surpass existing antibody-drug conjugate (ADC) therapies in tumor treatment.

[0344]

[0345] conclusion

[0346] Since CTPin, which targets CP2c, was identified as a cell-penetrating anticancer peptide, the inventors have sought ways to apply it more effectively to MTA therapy. Consequently, the inventors established a dual-targeting anticancer therapeutics (DTAT) platform as a molecular targeted therapy (MTA) capable of targeting both extracellular tumor-specific antigens (TSAs) and intracellular CP2c. The emergence of drug resistance is recognized as a potential drawback of MTA therapy (Ref. 1). However, the anticancer cytotoxicity of CPTin is unrelated to known mutations that cause drug resistance (Ref. 13).

[0347] The trastuzumab-CPTin of the present invention utilized the whole antibody trastuzumab as the carrier of the CPTin payload. Despite potential benefits such as low-cost production and high tumor penetration efficiency, AbFs lacking the constant fragment (Fc) containing scFv have inherent disadvantages in that they are rapidly eliminated from the blood and cannot trigger Fc-mediated effector functions (References 28-30). Indeed, compared to trastuzumab, DTAT-D311 showed a poor in vivo pharmacokinetic profile, while its high efficacy was attributed to the potent cytotoxicity of CPTin.

[0348] The trastuzumab-CPTin of the present invention can carry 2 to 8 CPTin molecules, whereas DTAT-D311 can contain only a single payload. In addition, the pan-cancer cytotoxicity of CPTin was confirmed in DTAT4-Tra, which suggests the valuable utility of CPTin as a universal cytotoxic payload loaded on antibodies for targeted anticancer immunotherapy.

[0349] Furthermore, CPTin possesses advantageous properties compared to existing chemicals used in the production of antibody-drug conjugates (ADCs), in that it is a normal peptide and selectively exhibits cytotoxicity toward cancer cells. Therefore, DTATs are expected to overcome the manufacturing complexity and clinical safety issues associated with ADCs. In the former case, the ADC manufacturing process following antibody production essentially involves separate chemical reactions for the conjugation of the linker and payload, followed by subsequent processes (References 16 and 17). In contrast, DTATs represented by the trastuzumab-CPTin of the present invention utilize common peptides for both the CS linker and the payload.

[0350] Consequently, since DTAT is a single protein molecule that does not require non-protein components, it can be simply produced using existing recombinant technology. Such a simple manufacturing process is economically advantageous as it can lower production costs. In addition, the production yield of trastuzumab-CPTin was much higher than that of the parent molecule, trastuzumab.

[0351] Regarding safety issues, dose-limiting toxicity (DLT) is often shared among different ADCs with the same cytotoxic chemical payload, so ADC doses are limited to levels lower than those required for optimal anticancer efficacy (Ref. 32). On the other hand, as expected from the cancer cell-selective cytotoxicity of the CPTin peptide payload, DTAT4-Tra did not exhibit significant toxicity in vivo even at high doses.

[0352] Furthermore, since both trastuzumab and CPTin are unlikely to exhibit immunogenicity in humans, a much higher maximum tolerated dose (MTD) with sufficient efficacy is reasonably expected for the clinical use of DTAT. In this regard, the fact that DTAT4-Tra administered at non-toxic high doses achieved higher efficacy along with faster tumor regression than trastuzumab-emtansine (Kadcyla®) and the recently developed anti-HER2 ADC trastuzumab-deruxtecan (Enhertu®) administered at clinical doses implies that DTAT may be superior to ADCs in terms of in vivo effects when considering both safety and efficacy.

[0353]

[0354] As demonstrated in the trastuzumab-CPTin of the present invention, DTATs produced by single recombinant production have an invariant DAR with identical CPTin junction sites. In contrast, early generation ADCs were prepared as heterogeneous mixtures (Ref. 31). For example, trastuzumab-emtansine products contain 0 to 8 emtansine molecules with an average DAR of 3.5, in which linker-payload chemicals are nonspecifically attached to lysine residues of trastuzumab (Ref. 33 and 34).

[0355] However, since homogeneity of the DAR and the binding site is desirable for the optimal efficacy and safety of ADCs, there is an increasing demand to produce homogeneous products with precise drug loading and predetermined, controlled binding sites (Ref. 31). Accordingly, the recently developed trastuzumab-deruxtecan has a nearly homogeneous DAR 7-8 by attaching the linker-payload to the free cysteine ​​of trastuzumab (Ref. 35 and 36). In DTAT, the CPTin payload peptide is linked to the C-terminus of the polypeptide chain. The present invention demonstrates that both DTAT2-Tra and DTAT4-Tra can be stably produced without loss of the CPTin payload and maintain stability in serum. Thus, another advantage of using the CPTin payload is that perfect homogeneity and uniformity of the DTAT product can be achieved.

[0356]

[0357]

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[0398]

[0399] II. DTAT-Cet

[0400] 실시예: EGFR 표적 항체를 포함하는 DTAT 플랫폼 (DTAT2 / 4 / 8-Cet)

[0401] The inventors investigated the anticancer activity of a fusion protein comprising a tumor-targeting antibody, an MMP cleavage sequence, an anticancer peptide, and a cell-penetrating peptide, and confirmed that the anticancer effect was most superior when using a specific combination of MMP cleavage sequence, anticancer peptide, and cell-penetrating peptide (scFv / antibody-MMP-COT-iRGD). Accordingly, the fusion protein of the present invention was named the DTAT (Dual-Targeting Antibody-based Therapeutics) platform. This name was devised because the tumor is targeted primarily by a tumor target delivery vehicle, and the tumor is targeted secondarily by a cell-penetrating anticancer peptide that targets CP2c.

[0402]

[0403]

[0404] Experimental method

[0405] 1. Reagents

[0406] pcDNA for protein expression TM 3.4 TOPO TM The plasmid vector was purchased from Invitrogen (#A14697, Carlsbad, CA, USA). Recombinant DNA containing chemically synthesized oligonucleotides was purchased from Bioneer (Daejeon, Korea), amplified by PCR, and inserted into the plasmid vector.

[0407] Transfection for DTATs-Cet expression is ExpiFectamine TMThe test was performed using the CHO Transfection Kit (Thermo Fisher, MA, USA). Horseradish peroxidase (HRP)-conjugated protein-A for ELISA was purchased from Cell Signaling Technology (#12291; Danvers, Massachusetts, USA), and Human EGF R protein, His tag (EGR-H5222), and FITC-labeled protein-L (#RPL-PF141) for flow cytometry were purchased from ACROBiosystems (Seoul, Korea). FQI1 (SML0413) and cisplatin (P4394) were purchased from Sigma-Aldrich (St. Louis, MO).

[0408] Gemcitabine was purchased from Supelco (Phr2582, Bellefonte, PA, USA). CPTin was produced from E. coli BL21(DE3) strain. All reagents were diluted to experimental concentrations in phosphate-buffered saline (PBS) and used for in vitro and in vivo experiments. Target proteins were analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Each sample was uniformly loaded, and the gel was stained with Coomassie Brilliant Blue G-250 (B7920, Sigma-Aldrich, St. Louis, MO).

[0409] Female BALB / c nude mice (5 weeks old) weighing 13–16 g were purchased from Orient Bio (Seongnam, Korea). Mice were housed under standard conditions (22±2℃, 55±5% humidity, 12-hour / 12-hour light-dark cycle), and food and water were provided free of charge. All experiments were approved and performed in accordance with the guidelines of the Konkuk University Animal Ethics Committee (#KU23263).

[0410]

[0411] 2. Cell line

[0412] AsPC1, BxPC3, Capan1, Capan2, MIA PaCa2, Panc1 and SNU213 (pancreatic cancer), SKOV3 (ovarian cancer), MCF3 and SKBR3 (breast cancer), MDA-MB-231 and MDA-MB-468 (triple-negative breast cancer), A431 and A2058 (skin cancer), SNU1041 (head and neck cancer), SW480 and HCT116 (colorectal cancer), HT29, A549, H441 and H460 (lung cancer), THP-1 and Jurkat (blood cancer), and hMSC (stem cell) cell lines were purchased from the Korean Cell Line Bank (KCLB, Seoul, South Korea). Cultures were performed at 37°C and 5% CO2 in high-glucose Dulbecco's modified Eagle medium or Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 μg / mL). ExpiCHO-S cells were purchased from Thermo Fisher (#A29127) and cultured in ExpiCHO expression medium (Thermo Fisher, #A2910001) in an 8% CO2 incubator maintained at 37°C and 110 rpm humidity. Cells were cultured 3–4 x 10⁻¹⁰ one day prior to transfection. 6 They were dispensed at a density of cells / mL. After 24 hours, the cell stock was 7–10×10 6 A density of cells / mL was reached, and then new medium was added for 6×10 6 It was diluted to cells / mL. Mouse embryonic fibroblasts (MEF) were isolated from pregnant CD-1 mice (Orient Bio, Seongnam, Korea) and cultured in high-glucose DMEM supplemented with 10% FBS at 37°C and 5% CO2.

[0413]

[0414] 3. Design and Preparation of Expression Vector for DTATs-Cet

[0415] To develop the DTAT-D351 and DTATs-Cet plasmids, chemically synthesized and codon-optimized recombinant sequences were processed through the XbaI and NotI restriction enzyme sites of pcDNA3.4 TM It was inserted into the TOPO vector. The sequences used are as follows, and CPTin (CP2c targeting inhibitor) consists of a CP2c oligomerization domain targeting motif (COT) and a cell permeation peptide (iRGD) that target CP2c.

[0416] DTAT-D351: [cetuximab heavy chain variable region]-[GGGGS triple repeat linker]-[cetuximab light chain variable region]-[MMP11 CLS]-[CPTin]

[0417] DTAT-Cet HC: [Cetuximab heavy chain]-[MMP11 CLS]-[CPTin]

[0418] DTAT-Cet LC: [Cetuximab light chain]-[MMP11 CLS]-[CPTin]

[0419] In addition, HER1 scFv and cetuximab plasmids were constructed by inserting a sequence with [MMP11 CLS]-[CPTin] removed.

[0420] To induce point mutations in the DTAT-Cet light chain, site-specific mutagenesis using synthetic primers was performed to construct the DTAT-Cet LC mutant MMP11 CLS and DTAT-Cet LC mutant iRGD plasmids. Additionally, for the DTAT-Cet LC mutant COT, a chemically synthesized DNA fragment containing the desired mutation was inserted into the DTAT-Cet light chain plasmid using the BamHI and NotI restriction enzyme sites. All recombinant DNA constructs were verified by DNA sequencing.

[0421] Meanwhile, DTAT8-Cet was bound to the ends of the light and heavy chains of cetuximab, respectively, with [MMP11 CLS]-[COT]-[linker (sequence number 12)]-[CPTin] ("H++" and "L++" in Fig. 11A).

[0422]

[0423] The protein was expressed by transfecting CHO-S cells and MMP11 KO CHO-S cells, and the transfection was performed using ExpiFectamine TM It was performed according to the manufacturer's protocol for the CHO Transfection Kit (Thermo Fisher). Briefly, 6X10 6 CHO-S cells were transfected with plasmids encoding recombinant proteins. For the expression of all antibodies except DTAT-D351, light-chain and heavy-chain plasmids were transfected in a 1:1 ratio. For transfection, the plasmid DNA and transfection reagents were each diluted in OptiPRO SFM (Thermo Fisher), immediately mixed, and incubated for 5 minutes. The resulting transfection mixture was applied directly to CHO-S cells. After 18–20 hours, an enhancer, a feed solution, and 20% glycerol were added to the culture medium to improve protein expression and stability.

[0424] After 5 days, cells were harvested, and the culture supernatant was collected for protein purification. Recombinant proteins were purified using FPLC affinity chromatography with a HiTrap Protein A HP column (Cytiva Korea, Incheon, Korea). DTAT-D351 was HisPur TMIt was purified using Ni-NTA resin (88222, Thermo Fisher, MA, USA). The concentration of the purified protein was precisely quantified by spectrophotometry at 280 nm using the molar absorbance value calculated from the amino acid sequence using the ProtParam tool (https: / web.expasy.org / protparam / ).

[0425]

[0426] Information on each antibody and sequence used in this invention is summarized in the table below.

[0427] 이이서열번호HER1 scFv(아미노산)LEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGG SQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAGS25Cetuximab VH(아미노산)LEQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA26Cetuximab Light chain(아미노산)LEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGS27CetuximabH-MMP11-COT-iRGD(amino acid)LEQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPKGSGGYAELRMGGNYPQRPCRGDKGPDC28Cetuximab L-MMP11-COT-iRGD(amino acid)LEDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGGYAELRMGGNYPQRPCRGDKGPDC29

[0428]

[0429] 서열서열번호HER1 scFv(염기)CTCGAGGATATATTATTAACGCAGAGTCCAGTCATCCTGTCAGTTTCACCTGGTGAACGTGTTAGTTTCAGTTGCCGTGCATCCCAATCCATTGGAACTAATATCCACTGGTATCAGCAGAGAACTAATGGCAGTCCAAGGTTACTAATAAAGTACGCTTCAGAATCTATCTCCGGCATACCTTCCAGATTTTCTGGTTCTGGTTCTGGAACGGACTTTACCCTTTCTATTAATTCTGTAGAGTCCGAAGATATTGCCGATTATTACTGTCAACAAAATAATAACTGGCCCACCACGTTCGGCGCCGGTACCAAGTTAGAACTAAAAGGTGGTGGCGGTAGTGGTGGTGGCGGTTCTGGAGGCGGAGGCTCACAGGTGCAGCTGAAACAGTCCGGCCCAGGACTAGTTCAACCATCCCAATCACTTTCAATAACCTGTACTGTATCAGGATTCTCATTAACCAACTACGGCGTCCACTGGGTCCGTCAGAGTCCCGGTAAAGGCTTGGAATGGCTAGGCGTAATATGGAGTGGCGGTAACACAGACTACAATACCCCCTTTACATCCCGTTTAAGTATTAATAAAGATAATTCAAAGTCTCAAGTCTTCTTTAAAATGAACTCCCTTCAGTCCAATGATACAGCAATCTACTACTGCGCTAGGGCTCTTACATATTATGACTATGAATTTGCATACTGGGGTCAGGGAACTCTGGTAACAGTAAGTGCTGGATCC30CetuximabVH(염기)CTCGAGCAGGTGCAGCTGAAGCAGTCTGGCCCCGGCTTGGTCCAGCCTAGCCAGTCCCTGTCCATCACCTGTACCGTGTCTGGATTCAGCCTGACCAACTACGGCGTGCACTGGGTGCGGCAGAGCCCTGGCAAGGGCCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAATACCGACTACAACACACCTTTTACCAGCAGACTGTCTATCAACAAGGACAACAGCAAATCCCAAGTGTTCTTCAAGATGAACTCCCTGCAAAGCAACGACACCGCCATCTACTACTGCGCCAGAGCCCTGACCTACTATGATTACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCGCT31Cetuximab lightchain(염기)CTCGAGGACATCCTGCTGACCCAATCTCCCGTCATCCTGTCTGTGTCTCCTGGCGAGAGAGTGTCTTTCTCTTGCAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAATGGCTCGCCTAGACTGCTGATCAAGTACGCCAGCGAGTCTATTTCTGGAATCCCCAGCCGGTTCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGAGCATCAACTCCGTCGAGAGCGAAGACATCGCCGACTACTACTGCCAGCAGAACAACAATTGGCCTACCACATTTGGAGCTGGCACCAAGCTGGAACTGAAGCGGACCGTGGCCGCTCCTAGCGTGTTCATCTTCCCTCCAAGCGACGAGCAGCTGAAATCCGGCACAGCTTCTGTGGTGTGCCTGTTGAACAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCCCTGCAAAGCGGCAACAGCCAAGAGTCCGTGACCGAACAGGACAGCAAGGATTCCACCTACTCTCTGAGCAGCACCCTCACACTGTCCAAGGCCGATTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACACACCAGGGCCTGTCCTCCCCTGTGACAAAGAGCTTCAACAGAGGCGAATGTGGATCC32CetuximabH-MMP11-COT-iRGD(염기)CCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGATCCGGTGGTTATGCAGAACTGCGTATGGGCGGCAACTATCCGCAGCGTCCGTGTCGTGGTGATAAAGGTCCGGATTGC33CetuximabL-MMP11-COT-iRGD(염기)CTCGAGGACATCCTGCTGACCCAATCTCCCGTCATCCTGTCTGTGTCTCCTGGCGAGAGAGTGTCTTTCTCTTGCAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAATGGCTCGCCTAGACTGCTGATCAAGTACGCCAGCGAGTCTATTTCTGGAATCCCCAGCCGGTTCTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGAGCATCAACTCCGTCGAGAGCGAAGACATCGCCGACTACTACTGCCAGCAGAACAACAATTGGCCTACCACATTTGGAGCTGGCACCAAGCTGGAACTGAAGCGGACCGTGGCCGCTCCTAGCGTGTTCATCTTCCCTCCAAGCGACGAGCAGCTGAAATCCGGCACAGCTTCTGTGGTGTGCCTGTTGAACAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCCCTGCAAAGCGGCAACAGCCAAGAGTCCGTGACCGAACAGGACAGCAAGGATTCCACCTACTCTCTGAGCAGCACCCTCACACTGTCCAAGGCCGATTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACACACCAGGGCCTGTCCTCCCCTGTGACAAAGAGCTTCAACAGAGGCGAATGTGGATCCGGTGGTTATGCAGAACTGCGTATGGGCGGCAACTATCCGCAGCGTCCGTGTCGTGGTGATAAAGGTCCGGATTGC34

[0430]

[0431] 4. 인 비트로 세포독성 실험

[0432] For the cytotoxicity test, cells were plated in 90 μL of medium in 96-well plates at a cell density of approximately 5,000 cells per well. The plates were incubated for 24 hours at 37°C in a 5% CO2 humidified environment. After incubation, 10 μL of test samples, prepared by dissolving the recombinant protein and various drug formulations in PBS, were added to each well. Each compound was prepared at its maximum concentration, and then the IC50 50 Serial dilution was performed to generate a concentration-response curve for measurement. After culturing the cells for an additional 24 hours, 10 μL of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader (BioTek, Vermont, USA) to evaluate cell viability.

[0433]

[0434] 5. ELISA

[0435] For the immunoassay of HER1 scFv, cetuximab, and DTATs-Cet, 0.5 μg / mL of EGFR His (ACROBiosystems) was coated onto 96-well immunoassay plates and incubated overnight at 4°C. The EGFR His was diluted in 100 mM bicarbonate / carbonate coating buffer (pH 9.6). To block non-specific binding, 200 μL of 1% BSA dissolved in PBS was added to each well and incubated at 25°C for 1 hour. After blocking binding, protein samples were added to the wells and incubated at 25°C for 1 hour. Next, 100 μL of Protein A-HRP (Cell Signaling Technology) diluted 1:10,000 was added to each well and incubated for another hour at 25°C. After incubation, 100 μL of TMB substrate solution (Thermo Scientific) was added, and the reaction was carried out at 25°C for 20 minutes. To stop the reaction, 100 μL of 1N HCl was added to each well. Absorbance was measured at 450 nm using a microplate reader (BioTek). To ensure accuracy, the immunoplate was washed three times with PBS-T (pH 7.4) throughout the entire process, including the coating, blocking, binding, detection, and reaction steps.

[0436]

[0437] 6. Flow cytometry

[0438] The antigen binding ability of protein samples on the cell surface was evaluated using flow cytometry. For the analysis, A431 cells (approximately 10 6Cells were cultured with various concentrations of each protein sample (0.1 pM–400 nM) at 4°C for 30 minutes. After culture, cells were washed twice with flow cytometry staining buffer (1% BSA / PBS) to remove unbound proteins. To detect DTATs-Cet and cetuximab binding, cells were cultured for an additional 90 minutes with FITC-labeled protein L 5 μg / mL. After washing twice more with staining buffer, quantification was performed by flow cytometry using a Novocyte instrument (Agilent, CA, USA).

[0439]

[0440] 7. Mass Spectrometry

[0441] The precise molecular weight of the purified protein was measured using a MALDI-TOF mass spectrometer (Ultraflextreme, Bruker Daltonics, Bremen, Germany) operating in linear mode with a 337 nm smart beam laser. Prior to analysis, the protein sample was prepared by mixing a sinapinic acid matrix with a 70% (v / v) acetonitrile / H2O solution containing 0.1% trifluoroacetic acid.

[0442]

[0443] 8. Propidium iodide (PI) staining

[0444] To investigate the process of apoptosis over time in A431 cells, a staining method using propidium iodide (PI) was used. First, 5,000 cells (80 μL of medium) were seeded per well in a 96-well plate and cultured at 37°C for 24 hours. Subsequently, 10 μL of PI solution (10 μg / mL) and 10 μL of drug solution (protein drug 25 μM, chemical drug 250 μM) were added to each well, and the cells were cultured for up to 24 hours while observing at set time intervals using a fluorescence microscope.

[0445]

[0446] 9. Stability Test

[0447] To investigate the stability of cetuximab and DTAT4-Cet, proteins were prepared at planned assay concentrations in buffer (20% glycerol in PBS) or human serum and dispensed into separate tubes for incubation at 4°C, 24°C (room temperature), or 37°C. For SDS-PAGE analysis, human serum was stripped with IgG using a HiTrap Protein A HP column (Cytiva Korea). At specified time points (days 0, 1, 2, 4, 8, and 32), each sample was flash-frozen in liquid nitrogen (LN₂) and stored at -80°C. Subsequently, SDS-PAGE, ELISA, and CCK-8 assays were performed according to the previously described protocol, maintaining a maximum CCK-8 treatment concentration of 10 μM.

[0448]

[0449] 10. Single-dose toxicity test

[0450] Acute toxicity studies were conducted in accordance with the guidelines of the Organization for Economic Cooperation and Development (OECD). Female mice were weighed and randomly divided into four groups: 6 mice per group for the experimental group and 4 mice per group for the vehicle. After fasting overnight, each group was administered a single intravenous injection of the drug at doses of 0, 100, or 300 mg / kg (mpk). Signs of toxicity were monitored for two days. Water and feed were provided on a free basis, and the daily body weight and feed intake of each mouse were recorded. After the two-day observation period, the mice were sacrificed, their organs were excised and weighed, and blood samples were collected for complete blood count (CBC) and differential analysis.

[0451]

[0452] 11. Research on In Vivo Xenograft Mouse Models

[0453] The xenograft mouse model consists of 5x10 female BALB / c nude mice. 6 M16 MDA-MB-468 cells were injected subcutaneously to establish the tumor. When the average tumor volume reached approximately 150 mm³, mice were randomly assigned to each group. Mice were intravenously administered a designated dose of the drug every 3 days (Q3D) for a total of 8 treatments. Tumor size and body weight were recorded every 3 days. Three days after the last (8th) treatment, mice were euthanized to evaluate the residual drug effect, and tumors were harvested and weighed.

[0454]

[0455] 12. Statistical Processing

[0456] Statistical analysis was performed using GraphPad Prism version 8.0 software (www.graphpad.com, USA) by applying appropriate statistical methods. Data were presented as mean ± standard deviation (SD) for in vitro experiments and mean ± standard error (SEM) for in vivo experiments. The concentration required for 50% inhibition (IC10)50 ) and dissociation constant (K d ) was determined using the nonlinear least squares method. Statistical significance was evaluated using p-values. Not significant: p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0457]

[0458]

[0459]

[0460] Experimental results

[0461] 1. Design and Production of DTAT-D351

[0462] Before applying the DTAT platform to cetuximab, the DTAT platform was validated in the form of a single-strand variable fragment (scFv). The heavy-strand variable region (VH) and light-strand variable region (VL) of cetuximab were linked using a GGGGS triple repeat linker, and the insert was inserted into the pcDNA 3.4 plasmid via XbaI and NotI. The cetuximab scFv produced in this way was named HER1 scFv. Subsequently, MMP11 CLS and CPTin, which are DTAT platform components of the HER1 scFv, were combined and inserted into the plasmid as described above, and named DTAT-D351 (Fig. 8A).

[0463] After transfecting CHO-S cells with the constructed plasmid and expressing it for 5 days, HiTrap in the culture medium TM Purification was performed using MabSelect and verified by SDS-PAGE (Fig. 8B). The relative production yield was calculated using ImageJ, taking into account the mass of the image in Fig. 8B. DTAT-D351 showed a production yield of 90% compared to HER1 scFv (Fig. 8C).

[0464]

[0465] The produced HER1 scFv and DTAT-D351 were treated to the EGFR-overexpressing epithelial cell carcinoma cell line A431 at a maximum concentration of 10 μM for 24 hours, and cell viability was compared to that of the control drug cisplatin (maximum 100 μM) using CCK8 assay. As a result, DTAT-D351 had an IC50 of 2.82 μM. 50 and an IC of 4.66 μM 80 It showed approximately 8 times (IC) compared to the control drug cisplatin. 50 : 24.95 μM) and 9 times (IC 80 : 42.68 μM) was high (Fig. 8D). As a result of applying the DTAT platform, it was found that although the protein production yield decreased slightly, it showed superior efficacy compared to cisplatin.

[0466]

[0467] 2. Verification of DTAT-D351 functions

[0468] The effect of applying the DTAT platform on the target binding affinity of HER1 scFv was confirmed through enzyme immunosorbent assay (ELISA) and flow cytometry.

[0469] The target binding affinity of HER1 scFv and DTAT-351 for EGFR was measured via ELISA. K for EGFR d The values ​​were similar, at 0.990 nM for HER1 scFv and 1.047 nM for DTAT-D351 (Fig. 9A).

[0470] Flow cytometry was performed using A431 cells, which are EGFR-overexpressing cells, to confirm target affinity for cell surface antigens. A431's K d The values ​​were 17.78 nM for HER1 scFv and 17.98 nM for DTAT-D351, which were similar to the ELISA results (Fig. 9B). These results show that the application of the DTAT platform does not affect the target affinity of HER1 scFv.

[0471]

[0472] 3. DTAT-Cet Design and Production

[0473] In the production process of DTATs-Cet, the DTAT platform using a cetuximab full antibody exhibited a problem where cleavage linker sequences (CLS) were partially cleaved from the heavy chain during expression (Fig. 10B). Accordingly, in the CHO-S expressing cell line, the complete form of DTAT2 / 4-Cet was produced by adding 20% ​​glycerol during cell culture (Fig. 10C).

[0474]

[0475] ELISA and flow cytometry analysis showed that the binding affinities of HER1 scFv and DTAT-D351 were similar but lower than those of the whole antibody cetuximab. Additionally, scFvs tend to have shorter half-lives than whole antibodies. Therefore, DTAT was produced using a whole antibody. Specifically, DTAT2 / 4 / 8-Cet was produced in CHO-S cells using cetuximab, a tumor-targeting antibody that targets EGFR (Epidermal Growth Factor Receptor).

[0476] Cetuximab heavy chain (mAb-Cet HC), DTAT-cetuximab heavy chain (DTAT-Cet HC), cetuximab light chain (mAb-Cet LC), and cetuximab light chain (DTAT-Cet LC) were inserted into pcDNA 3.4 plasmid via Xba° and Not° (Fig. 11A).

[0477]

[0478] The constructed plasmid was expressed in glycerol-supplemented medium for 5 days via co-transfection combining the heavy and light chains in CHO cells, and then HiTrap TMThe samples were purified in the culture medium using MabSelect and verified by SDS-PAGE (Figs. 11B and 11C). The cetuximab modified with DTAT was subsequently named DTATs-Cet. Production yield was expressed in mg per 1 L of culture medium. mAb-Cet showed a production yield of 163.8 mg, DTAT2L-Cet (two anticancer modules bound to the light chain) 175.4 mg, DTAT2H-Cet (two anticancer modules bound to the heavy chain) 176.1 mg, DTAT4-Cet 185.9 mg, and DTAT8-Cet 215.1 mg (Fig. 11D).

[0479]

[0480] DTATs-Cet was treated to A431 cells at a maximum concentration of 10 μM for 24 hours, and cell viability was compared to that of the control drug cisplatin (maximum 100 μM) via CCK8 analysis.

[0481] As a result, IC 50 The levels were about 20 times higher (1.22 μM) for DTAT2L-Cet, about 19 times higher (1.27 μM) for DTAT2H-Cet, about 29 times higher (0.84 μM) for DTAT4-Cet, and about 54 times higher (0.46 μM) for DTAT8-Cet compared to cisplatin (24.95 μM).

[0482] IC 80 The levels were approximately 14 times higher (2.85 μM) for DTAT2L-Cet, approximately 14 times higher (2.92 μM) for DTAT2H-Cet, approximately 25 times higher (1.69 μM) for DTAT4-Cet, and approximately 47 times higher (0.89 μM) for DTAT8-Cet compared to cisplatin (42.68 μM) (Fig. 11E).

[0483] It was found that applying the DTAT platform slightly increased protein production yield and showed superior efficacy compared to cisplatin.

[0484]

[0485] The molecular weights of the produced DTAT2-Cet and DTAT4-Cet were analyzed using MALDI TOF. As shown in Figure 11E, there was no significant difference in the relative cell viability between DTAT2L-Cet and DTAT2H-Cet, so only DTAT2L-Cet and DTAT4-Cet were analyzed.

[0486] As a result, DTAT2L-Cet was measured at 156.0 kDa, and DTAT4-Cet was measured at a predicted mass of 162.5 kDa (Figs. 12A and 12B). These results demonstrate that DTAT2L-Cet and DTAT4-Cet were fully expressed and purified without any sequence omissions. Furthermore, the mass spectrometry results showed a single peak at the predicted molecular weight, indicating that the intact form of DTAT2 / 4-Cet was produced. In contrast to ADCs where the DAR does not fall into an integer ratio due to heterogeneous antibody-drug binding, the single peak of DTAT2 / 4-Cet confirmed that the DAR is constant.

[0487] In addition, the relative affinity of DTAT2 / 4-Cet for EGFR1 protein or cell line A431 overexpressing EGFR1 was analyzed by ELISA and FACS methods. The results of the affinity analysis confirmed that the change in affinity of DTAT2 / 4-Cet compared to the monoclonal antibody was minimal (Figs. 12C and 12D).

[0488] These results indicate that the application of the DTAT_platform source technology (CLS-COT-CPP) hardly reduces the affinity for the EGFR1 protein molecule and the EGFR1 overexpressing cell line A431.

[0489]

[0490] 4. Confirmation of Anticancer Activity of DTAT2 / 4-Cet-1

[0491] The anticancer activity of the produced DTAT2 / 4 / 8-Cet was confirmed in various tumor cell lines, immune cell lines, and normal cell lines.

[0492] As a result, it was confirmed that DTAT2 / 4 / 8-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. 13).

[0493] In addition, anticancer activity increased in the order of DTAT-D351 (DTAT platform applied to Cet scFv), DTAT2-Cet, DTAT4-Cet, and DTAT8-Cet, and it was confirmed that anticancer activity increased as DAR increased (Fig. 13).

[0494]

[0495] To determine the effect of each module of the DTAT platform on the efficacy of the existing DTAT-Cet, point mutations were introduced into each module. The mutation sequences for each module were L242D for the CLS mutation, D258A for the CPP mutation, and Q250N for the COT mutation. These mutation sequences were applied to DTAT-Cet LC and expressed together with mAb-Cet HC to generate mDTAT2L-Cet (DTAT2L-Cet CLS mutation, DTAT2L-Cet COT mutation, DTAT2L-Cet CPP mutation) (Fig. 14A).

[0496] The generated and purified mDTAT2L-Cet was verified by SDS-PAGE together with the control DTAT2L-Cet (Fig. 14B).

[0497] In addition, A431 cells were treated with each mutant, and relative cell viability was compared to the control group, DTAT2L-Cet WT, via CCK8 analysis after 24 hours. DTAT2L-Cet is IC 50 This was found to be 1.23 μM, which reduced cell viability, but the mutated mDTAT2L-Cet construct did not affect cell viability (Fig. 14C). These results demonstrate that each module of the DTAT platform is essential for efficacy.

[0498]

[0499] Considering the results to date, the anticancer activity of DTAT-Cet is achieved through the following mechanism (Fig. 15): 1) cetuximab binds to EGFR to target tumor cells; 2) CLS is cleaved, leading to the cleavage of CPTin; 3) CPTin enters the tumor cell; and 4) CPTin targets CP2c, an oncogene, which induces apoptosis. 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-Cet exhibits excellent tumor specificity and safety for normal cells.

[0500]

[0501] 5. Stability of DTATs-Cet

[0502] Next, the stability of DTATs-Cet was evaluated. First, fluorescence and static light scattering with respect to temperature changes at 266 nm (SLS266) for mAb-Cet, DTAT2L-Cet (a form in which an anticancer peptide is attached to the light chain of a cetuximab antibody on the DTAT platform), and DTAT4-Cet were measured using the Uncle system, and hydrodynamic diameters were analyzed by measuring dynamic light scattering (DLS) at 25°C. The analysis results showed no significant difference in fluorescence with respect to temperature changes between Erbitux, DTAT2L-Cet, and DTAT4-Cet (Fig. 16A).

[0503] Both samples showed similar patterns with Erbitux in SLS266 (Fig. 16B). Similar hydrodynamic diameters were also confirmed in DLS analysis at 25°C (Fig. 16C).

[0504] Afterwards, the stability of DTAT4-Cet relative to Erbitux was analyzed using ELISA, CCK8 assay, and SDS-PAGE in various buffers (phosphate-buffered saline (PBS) and human serum), temperatures (4℃, 24℃, 37℃), and times (0, 1, 2, 4, 8, 16, and 32 days).

[0505] For the ELISA, samples stored under protein storage conditions (PBS buffer and 4°C) and treatment conditions (human serum and 37°C) were used.

[0506] Both Erbitux and DTAT4-Cet maintained similar binding affinities over time under storage conditions. Erbitux (PBS, 4°C) was 0.12–0.13 nM, Erbitux (human serum, 37°C) was 0.12–0.13 nM, DTAT4-Cet (PBS, 4°C) was 0.36–0.39 nM, and DTAT4-Cet (human serum, 37°C) was 0.18–0.21 nM (Figs. 16D to 16G).

[0507] CCK8 analysis under identical storage conditions showed that both samples were unaffected by changes over time, and Erbitux was not detected regardless of the condition (ND). On the other hand, DTAT4-Cet (PBS, 4°C) maintained similar cell viability at 0.55–0.58 μM, and DTAT4-Cet (human serum, 37°C) maintained similar cell viability at 0.49–0.56 μM (Figs. 16H to 16K).

[0508] SDS-PAGE results confirmed that DTAT4-Cet stored in PBS was stable for up to 32 days at all test temperatures (Fig. 16L). DTAT4-Cet stored in human serum showed slight cleavage when tested at 24°C for 32 days, but remained stable for up to 32 days at 4°C and 37°C (Fig. 16M). Therefore, it was found that DTAT4-Cet exhibited stability similar to that of Erbitux.

[0509]

[0510] 6. Confirmation of Anticancer Activity of DTAT2 / 4 / 8-Cet-2

[0511] The cytotoxicity of DTAT2 / 4 / 8-Cet on anticancer cells was compared with other anticancer drugs.

[0512] For the cytotoxicity test, cells were plated in 90 μL of medium in 96-well plates at a cell density of approximately 5,000 cells per well. The plates were incubated for 24 hours at 37°C in a 5% CO2 humidified environment. After incubation, 10 μL of test samples, prepared by dissolving the recombinant protein and various drug formulations in PBS, were added to each well. Each compound was prepared at its maximum concentration, and then the IC50 50 Serial dilution was performed to generate a concentration-response curve for measurement. After culturing the cells for an additional 24 hours, 10 μL of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader (BioTek, Vermont, USA) to evaluate cell viability.

[0513] Specifically, CPTin, FQI1, cisplatin, gemcitabine, and lapatinib were treated to cancer cells (A431), immune cells (THP-1), and normal cells (MEF) in comparison to DTATs-Cet (Fig. 10).

[0514] DTAT-Cet, CPTin, and lapatinib were administered at a maximum of 10 μM, cisplatin and FQI1 at a maximum of 100 μM, and gemcitabine at a maximum of 10 mM.

[0515] In A431 cells, DTATs-Cet IC as DAR increases 50 These decreased to 2.21 μM, 1.02 μM, 0.61 μM, and 0.37 μM, respectively. The reference drug was the IC of DTAT4-Cet. 50Compared to (0.61 μM), FQI1 shows an IC difference of approximately 71 times (43.62 μM), cisplatin approximately 50 times (31.06 μM), and gemcitabine approximately 3400 times (2074 μM). 50 It was shown (Fig. 17A).

[0516] In addition, DTATs-Cet did not exhibit cytotoxicity in immune cells (THP-1) and normal cells (MEF), whereas other control drugs, including cisplatin, did exhibit cytotoxicity (Figs. 17B and 17C). These results suggest that the cytotoxicity of DTATs-Cet is specific to cancer cells and does not exhibit cytotoxicity in immune cells or cancer cells.

[0517]

[0518] 7. Confirmation of DTAT2-Cet's Induction of Cancer Cell Apoptosis

[0519] The CCK8 assay used above is a method for evaluating cell viability, so it is not suitable for determining whether DTATs-Cet induces apoptosis. Therefore, propidium iodide (PI) staining was used to determine whether DTATs-Cet induces apoptosis.

[0520] A431 cells were treated with cisplatin (25 μM), mAb-Cet (2.5 μM), DTAT2L-Cet (2.5 μM), and DTAT2L-Cet COT mutant (mDTAT2L-Cet) (2.5 μM) along with PI, and fluorescence images were taken using a fluorescence microscope at 0 min, 30 min, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours.

[0521] DTAT2L-Cet was stained by PI starting at 30 minutes, and the fluorescence intensity increased over time (Fig. 18A). Additionally, the fluorescence intensity at 24 hours was graphed. DTAT2L-Cet showed a fluorescence intensity of 19.48%, whereas NC (0.00%), cisplatin (0.99%), mAb-Cet (0.04%), and mDTAT2L-Cet (0.40%), which lost efficacy due to a COT point mutation, showed no or very low fluorescence intensity (Fig. 18B). These results indicate that DTAT2L-Cet induces apoptosis.

[0522]

[0523] 8. Functional verification of each module constituting the DTAT4-Cet single-dose toxicity assessment platform

[0524] Prior to in vivo experiments, a single-dose toxicity study was conducted to determine the upper limit of DTAT4-Cet administration. The experiment was performed using 5-week-old female Balb / c mice, with 4 mice in the vehicle group and 6 mice each in the Erbitux group (300 mg / kg), the DTAT4-Cet intermediate-dose group (100 mg / kg), and the DTAT4-Cet high-dose group (300 mg / kg). Mice were observed for 7 days prior to intravenous injection and sacrifice (Fig. 19A). Body weight and water and feed intake were monitored for 7 days after administration.

[0525] No significant change in body weight was observed during the 7 days of the experiment (Fig. 19B). Likewise, no significant change in water and feed intake was observed (Fig. 19C).

[0526] After sacrifice, organ weight, blood tests, and serum biochemical tests were performed.

[0527] The organs were separated to determine the weight of the ovaries, liver, spleen, kidneys, adrenal glands, thymus, lungs, heart, lymph nodes, brain, and pituitary gland. There was no significant difference in organ weight between the Erbitux administration group and the DTAT4-Cet administration group (Fig. 19D).

[0528] Blood tests were performed to check for red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean erythrocyte volume (MCV), mean erythrocyte hemoglobin (MCH), mean erythrocyte hemoglobin concentration (MCHC), platelet count (PLT), white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), and basophils (BASO). There were no specific significant results for DTAT4-Cet in the test results (Fig. 19E).

[0529] Serum chemistry tests were performed to check for alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total protein (TP), albumin (Alb), albumin / globulin (A / G), blood urea nitrogen (BUN), creatinine (Crea), and uric acid (UA). The results showed no specific changes in DTAT4-Cet (Fig. 19F).

[0530] In conclusion, no significant toxicity was confirmed with DTAT4-Cet.

[0531]

[0532] 9. Phosphoric in vivo anticancer activity of DTAT4-Cet

[0533] To evaluate the in vivo efficacy of DTAT4-Cet, experiments were conducted by xenografting MDA-MB-468, an EGFR-overexpressing cell, into immunodeficient mice. Administration was initiated when the tumor size reached an average of 150 mm³.

[0534] A total of 8 vaccinations were administered at 3-day intervals, and mice were sacrificed on day 25, 3 days after the final vaccination, and tumor weights were measured. The experimental groups consisted of 8 mice each of a mock group, an Erbitux group (137.2 nmol / kg, Q3D), and a DTAT4-Cet intermediate dose group (137.2 nmol / kg, Q3D) (Fig. 20A).

[0535] As a result of the experiment, the tumor growth inhibition rate calculated from the tumor growth rate at day 25 was superior in the DTAT4-Cet group (TGI 47.8%) compared to the Erbitux group (TGI 39.5%) (Figs. 20B and 20C).

[0536] After the experiment, the tumors were resected and weighed. The weight of the resected tumors showed a clear difference between the Mock group and each group, but there was no significant difference between Erbitux and DTAT4-Cet (Fig. 20D).

Claims

1. An antibody that specifically binds to Human Epidermal Growth Factor Receptor 2 (HER2); and A tumor dual-targeting fusion protein comprising one or more anticancer peptide modules connected to one end of the light chain and / or heavy chain of the above antibody, The above anticancer peptide module is MMP cleavage sequence represented by the amino acid sequence of SEQ ID NO. 7; An anticancer peptide represented by the amino acid sequence of SEQ ID NO. 8, linked to the C-terminus of the MMP cleavage sequence; and A tumor dual-target fusion protein in which a cell-permeating peptide represented by the amino acid sequence of SEQ ID NO. 9 is sequentially linked to the C-terminus of an anticancer peptide.

2. In paragraph 1, the tumor dual-target fusion protein, wherein the antibody specifically binding to human epidermal growth factor receptor 2 is a whole antibody.

3. In paragraph 2, the tumor dual-target fusion protein, wherein the full-length antibody specifically binding to human epidermal growth factor receptor 2 is trastuzumab, pertuzumab, or magetuximab.

4. In claim 1, the antibody and the anticancer peptide module are tumor dual-target fusion proteins having a drug-to-antibody ratio (DAR) of 1:1 to 10:

1.

5. In paragraph 4, the antibody and the anticancer peptide module are tumor dual-target fusion proteins having a drug-antibody ratio (DAR) of 2:1 to 8:

1.

6. An expression vector comprising a polynucleotide encoding a tumor dual-target fusion protein of any one of claims 1 to 5.

7. Method for producing a tumor dual-target fusion protein comprising the following steps: A step of transforming a host cell with the expression vector of claim 6; and A step of culturing host cells in a medium supplemented with 15 to 30% glycerol.

8. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, a tumor dual-target fusion protein of any one of claims 1 to 5 and / or an expression vector of claim 6.

9. A pharmaceutical composition for the prevention or treatment of cancer, wherein, in paragraph 8, the cancer is a human epidermal growth factor receptor 2 (HER2) positive cancer.

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