HER2-GST-SN-38 complex and pharmaceutical composition for preventing or treating proliferative diseases comprising same

Abstract

The present invention relates to a fusion protein comprising a glutathione-S-transferase and a protein having a target cell or target protein binding capacity, a pharmaceutical complex thereof and its use as a pharmaceutical composition. The fusion protein and the drug complex comprising the same according to one aspect can maintain a long residence time in vivo, and can be effectively delivered to a target cell due to improved targeting ability to the target cell, and thus can be effectively used as a targeted therapeutic agent.

Description

Technical Field

[0001] The invention relates to a fusion protein and its use as a drug carrier, a drug complex and a drug composition. The fusion protein comprises glutathione-S-transferase and a protein capable of binding to a target cell or a target protein. Background Art

[0002] Nanoparticles have excellent biodistribution capabilities and can regulate the degree of drug release, so they are used as useful tools in fields such as imaging devices and targeted therapeutic agents. It is well known that under normal circumstances, when the diameter of nanoparticles is 200nm, they can leak near the blood vessels around the tumor. Since there are no lymphatic vessels formed around the tumor, the pressure is low, so the leaked nanoparticles can continue to remain in the tumor tissue. This process is called the EPR effect (enhanced permeability and retention effect), through which the permeability and retention of drug carriers can be improved.

[0003] Representative examples of nanoparticles currently used commercially include paclitaxel (Abraxane) and doxorubicin (Doxil). However, vascular permeability varies from tumor to tumor, and the limitation is that when nanoparticles are injected intravenously, the mononuclear phagocyte system (MPS) can quickly accumulate in reticuloendothelial organs such as the liver and spleen in order to clear them. As a result, targeted therapeutic agents using nanoparticles can reduce the therapeutic effect, and side effects may occur due to the toxicity of nanoparticles.

[0004] In order to overcome these shortcomings, a method of encapsulating nanoparticles by poly(ethylene glycol)ylation (PEGylation) has been developed. When using these methods, the advantage is that the time for which nanoparticles circulate in the body's circulatory system can be extended, but on the contrary, the possibility of entering target cells is reduced and cells other than target cells are targeted through non-specific binding, which can also reduce the therapeutic efficiency.

[0005] When using nanoparticles as drug carriers, a strategy has been proposed to induce structural changes in nanoparticles to enhance the targeting ability of drug delivery and the EPR effect. Specifically, attempts have been made to improve the targeting ability of drug carriers by inducing structural changes, such as coating the surface of nanoparticles with antibodies, proteins or peptides that bind to receptors overexpressed in cancer cells. However, the limitation is that these methods do not effectively improve tumor targeting efficiency, but instead provide targeting ligands that enable nanoparticles to be cleared faster through the in vivo immune response of MPS, thereby not showing significant therapeutic effects in terms of overall tumor treatment effects.

[0006] Theoretically, when exposed to a physiological environment, the surface of the nanoparticle will naturally be surrounded by other biomolecules in a direction that reduces the surface energy, based on the combined effects of entropic arrangement of water molecules, charge compensation on the particle surface, or exposure of the hydrophobic part. At this time, various biomolecules are non-specifically adsorbed on the surface of the nanoparticle, a form called a protein corona. When the protein corona is surrounded by other molecules, its original molecular characteristics are changed, blocking the targeting ability to the target cell or organ and the various biological functions exhibited by the nanoparticle. Therefore, research is still ongoing on how to regulate the protein corona so that nanoparticles can be applied as targeted therapeutics at the clinical level.

[0007] Therefore, in the preparation of nanoparticle-based targeted therapeutics, a method of regulating it by first forming a protein corona on the surface of the nanoparticle has been developed. To this end, in order to avoid the blocking of the targeting ability by the protein corona, a method of minimizing the interaction with proteins in the serum by changing the surface of the nanoparticles to zwitterionic, PEG and carbohydrate residues, etc. has been disclosed. In addition, the nanoparticles can be pre-coated with dysopsonic proteins, and the protein corona can be regulated by the circulation of the desired protein in the plasma. Therefore, the nanoparticles pre-coated with the protein corona increase the stability of the colloidal state and have the effect of continuing the circulation time in the blood without being cleared by the MPS. However, even by these methods, the targeting ability of the target can be limited, and since the biochemical effects produced by the biological interaction between the protein used in the pre-coating and the nanoparticles are not clear, it is also limited in clinical application. Summary of the invention

[0008] Technical issues

[0009] On the one hand, a drug complex is provided, comprising: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2 (HER2); a linker connecting the glutathione-S-transferase and the antibody, the affibody or the diabody; and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and the glutathione (GSH) molecule.

[0010] On the other hand, a pharmaceutical composition for preventing or treating a proliferative disease is provided, comprising: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2 (HER2); a linker connecting the glutathione-S-transferase and the antibody, affibody or diabody; and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and the glutathione molecule.

[0011] On the other hand, a method for preventing or treating a proliferative disease is provided, the method comprising the following steps: administering an effective amount of a drug complex to an individual in need thereof, the drug complex comprising: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2 (HER2); a linker connecting the glutathione-S-transferase and the antibody, affibody or diabody; and 7-ethyl-10-hydroxycamptothecin bound to the glutathione-S-transferase and the glutathione molecule.

[0012] On the other hand, a use of a drug complex for preparing a pharmaceutical preparation for preventing or treating a proliferative disease is provided, wherein the drug complex comprises: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2 (HER2); a linker connecting the glutathione-S-transferase and the antibody, affibody or diabody; and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and the glutathione molecule.

[0013] Technical Solution

[0014] On the one hand, a drug complex is provided, comprising: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to HER2 (human epidermal growth factor receptor 2); a linker connecting the glutathione-S-transferase and the antibody, the affibody or the diabody; and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and the GSH (Glutathione) molecule.

[0015] In this specification, the term "antibody" is a term well known in the art, meaning a specific protein molecule indicated for an antigenic site. For the purpose of this specification, an antibody means an antibody that specifically binds to a target cell or a receptor expressed on a target cell, and these antibodies are prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and by a conventional method from the obtained protein. This also includes partial peptides that can be prepared from the protein, and the partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The form of the antibody of this specification is not particularly limited, as long as it is a polyclonal antibody, a monoclonal antibody, a humanized antibody or has antigen binding, then the antigen binding fragment as a part thereof is also included in the antibody of this specification, and may include all immunoglobulin antibodies.

[0016] In this specification, "affine molecule (affibody molecule)" may mean an antibody mimetic that can bind to a specific target protein (receptor). Typically, an affibody molecule consists of 20 to 150 amino acid residues and may consist of 2 to 10 alpha helices. In more detail, the affibody molecule may include an anti-ErbB affibody molecule (ab31889), a HER2-specific affibody molecule (ZHER2:342), an anti-EGFR affibody molecule (ZEGFR:2377), etc. And, it is not limited thereto, including all affibody molecules that can recognize a specific receptor or target protein in a cell. Examples of target receptors or target proteins that can be recognized by the affinity molecule can be: amyloid beta peptide, synuclein (e.g., α-synuclein), apolipoprotein (e.g., apolipoprotein A1), complement factor (e.g., C5), carbonic anhydrase (e.g., CAIX), interleukin-2 receptor alpha chain (IL2RA; CD25), cell surface CD antigen (e.g., CD28), or c-Jun, factor VIII, fibrinogen, GP120, H-Ras, Her2, Her3, HPV16 E7, IAPP (human islet amyloid polypeptide), immunoglobulin A (IgA), IgE, IgM, interleukins (e.g., IL-1, IL-6, IL-8, IL-17), insulin, Staphylococcal protein A domain (Staphylococcal protein A domain), Raf-1, LOV domain (Light-oxygen-voltage-sensing domain), or RSV G protein. Information about the affibody is described in Stefan Stahl et al., Affibody Molecules in Biotechnological and Medical Applications, Trends in Biotechnology, August 2017, Vol. 35, No. 8, the entire text of which is incorporated into this specification by reference.

[0017] In this specification, the term "linker" refers to a molecule that connects two or more chemical structures. Specifically, the linker can be a polypeptide consisting of 1 to 400, 1 to 200, or 2 to 200 arbitrary amino acids. The peptide linker can include Gly, Asn and Ser residues, and can also include neutral amino acids, such as Thr and Ala. Amino acid sequences suitable for peptide linkers are known in the art. Optimization of the linker can also be considered to adjust the copy number "n" to achieve appropriate separation between functional parts or maintain necessary interactions between internal parts (inter-moiety).

[0018] In one embodiment, the peptide linker can be a linker with protease resistance. Specifically, resistance means not being bound by proteases, and / or not being cleaved by proteases, and / or remaining stable when in contact with proteases, and / or retaining its activity.

[0019] In a specific example, the peptide linker can be a flexible linker comprising G, S and / or T residues. Other flexible linkers known in the art, such as G and S linkers, not only add polar amino acid residues to improve water solubility, but also add amino acid residues such as T and A to maintain flexibility. Specifically, the linker can have a general formula selected from (GpSs)n and (SpGs)n, in which case, independently, p is an integer from 1 to 10, s is 0 or an integer from 0 to 10, p+s is an integer less than 20, and n is an integer from 1 to 20. Examples of linkers are: (GGGGS)n (SEQ ID NO:2), (SGGGG)n (SEQ ID NO:3), (SRSSG)n (SEQ ID NO:4), (SGSSC)n (SEQ ID NO:5), (GKSSGSGSESKS)n (SEQ ID NO:6), (RPPPPC)n (SEQ ID NO:7), (SSPPPPC)n (SEQ ID NO:8), (GSTSGSGKSSEGKG)n (SEQ ID NO:9), (GSTSGSGKSSEGSGSTKG)n (SEQ ID NO:10), (GSTSGSGKPGSGEGSTKG)n (SEQ ID NO:11), or (EGKSSGSGSESKEF)n (SEQ ID NO:12), wherein n is an integer from 1 to 20, or from 1 to 10.

[0020] On the other hand, a polynucleotide is provided, which encodes a fusion protein that combines the GST molecule with an antibody, an affibody or a diabody molecule capable of binding to HER2.

[0021] In this specification, the term "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides in a single-stranded or double-stranded form. It includes RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and unless otherwise mentioned, also includes natural polynucleotides and their analogs with modified sugar or base sites. In a specific example, the polynucleotide is a short-chain polynucleotide.

[0022] In yet another aspect, a vector comprising the polynucleotide is provided.

[0023] In this specification, the term "vector" is a vector capable of expressing a target protein in a suitable host cell, and refers to a gene construct containing a regulatory element operably connected to express a gene insert. A vector according to an embodiment may include expression regulatory elements, such as a promoter, an operator, a start codon, a stop codon, a polyadenylation signal and / or an enhancer, and the promoter of the vector may be constitutive or inducible. In addition, the vector may be a vector for expression capable of stably expressing the fusion protein in a host cell. The vector for expression may be a conventional vector for expressing exogenous proteins in plants, animals or microorganisms in the art. The recombinant vector may be constructed by various methods known in the art. For example, the vector may include a selective marker for selecting a host cell containing the vector, and if the vector is reproducible, a replication origin may be included. In addition, the vector may be self-replicating or introduced into a host DNA, and the vector may be selected from the group consisting of a plasmid, a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, a herpes simplex virus and a vaccinia virus.

[0024] The vector comprises a promoter operable in animal cells, such as mammalian cells. According to one embodiment, suitable promoters include promoters derived from mammalian viruses and promoters derived from mammalian cell genomes, such as CMV (Cytomegalovirus) promoter, U6 promoter and H1 promoter, MLV (Murine Leukemia Virus), LTR (Long terminal repeat) promoter, adenovirus early promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 α promoter, metallothionein promoter, β-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, human phosphoglycerate kinase (PGK) promoter, mouse phosphoglycerate kinase (PGK) promoter and survivin promoter.

[0025] Furthermore, in the vector, the aforementioned fusion protein can be operably linked to a promoter. In this specification, the term "operably linked" refers to the functional binding between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or a transcriptional regulatory factor binding site array) and another nucleic acid sequence, whereby the regulatory sequence can regulate the transcription and / or translation of the other nucleic acid sequence.

[0026] Yet another aspect provides a host cell comprising the fusion protein, polynucleotide or vector.

[0027] The cell may be, for example, a eukaryotic cell may be a yeast, a mold, a protozoa, a plant, a higher plant and an insect, or an amphibian cell, or a mammalian cell such as CHO, HeLa, HEK293 and COS-1, etc.; for example, it may be a cultured cell (in vitro), a graft cell and a primary cell culture (in vitro and ex vivo), an in vivo cell, and a mammalian cell including a human being, which are commonly used in the art. In addition, the organism may be a yeast, a mold, a protozoa, a plant, a higher plant and an insect, an amphibian or a mammal. Furthermore, the cell may be an animal cell or a plant cell.

[0028] The types of active pharmaceutical ingredients delivered to an individual using a pharmaceutical carrier may include: anticancer agents, contrast agents (dyes), hormone agents, antihormonal agents, vitamin agents, calcium preparations, mineral preparations, carbohydrate agents, organic acid preparations, protein amino acid preparations, detoxifiers, enzyme preparations, metabolic preparations, diabetes combination agents, tissue regeneration drugs, chlorophyll preparations, dye preparations, tumor drugs, tumor therapeutic agents, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapeutic agents, antibiotic preparations, antiviral agents, complex antibiotic preparations, chemotherapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospira serum, blood preparations, biological preparations, analgesics, immunogenic molecules, antihistamines, allergy drugs, non-specific immunogenic preparations, anesthetics, stimulants, psychoneurological agents, small molecule compounds, nucleic acids, aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNA and microRNA, etc.

[0029] SN-38 can be an anticancer agent and may include a pharmaceutically acceptable salt thereof. "SN-38 (7-ethyl-10-hydroxycamptothecin)" is the active ingredient of irinotecan (also known as "CPT-11"), which exerts anti-tumor effects by inhibiting the activity of type I DNA topoisomerase. Compared with irinotecan, it can show cytotoxic activity up to 1,000 times higher against various cancer cells in vitro.

[0030] In the present invention, the binding of glutathione-S-transferase and SN-38 (7-ethyl-10-hydroxycamptothecin) can be carried out through GSH (glutathione). That is, the SN-38 can be GSH-bound SN-38. GSH can serve as a binding site for glutathione-S-transferase, connecting SN-38 to glutathione-S-transferase.

[0031] The SN-38 molecule can be a nanoparticle that carries SN-38 or is capable of carrying SN-38. According to the prior art, as long as the nanoparticle can be used as a drug carrier, the nanoparticle can be used without restriction. Specifically, it can be any one selected from the following group: mesoporous silica nanoparticles (MSN), gold nanoparticles, magnetic nanoparticles, nucleic acid-metal organic framework nanoparticles, and polymer nanoparticles. It can also be a particle with GSH (Glutathione) bound to the nanoparticle. Thus, the nanoparticle can be bound to a fusion protein containing GST.

[0032] In one specific example, the SN-38 molecule can be represented by the following chemical formula 1.

[0033] [Formula 1]

[0034]

[0035] On the other hand, a pharmaceutical composition for preventing or treating a proliferative disease is provided, the composition comprising: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2 (HER2); a linker connecting the glutathione-S-transferase and the antibody, the affibody or the diabody; and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and the glutathione (GSH) molecule.

[0036] On the other hand, a method for preventing or treating a proliferative disease is provided, the method comprising the following steps: administering an effective amount of a drug complex to an individual in need thereof, the drug complex comprising: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2 (HER2); a linker connecting the glutathione-S-transferase and the antibody, affibody or diabody; and 7-ethyl-10-hydroxycamptothecin bound to the glutathione-S-transferase and the GSH (glutathione) molecule.

[0037] On the other hand, a use of a drug complex for preparing a pharmaceutical preparation for preventing or treating a proliferative disease is provided, wherein the drug complex comprises: a glutathione-S-transferase (GST) molecule; an antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2 (HER2); a linker connecting the glutathione-S-transferase and the antibody, affibody or diabody; and 7-ethyl-10-hydroxycamptothecin bound to the glutathione-S-transferase and the GSH (glutathione) molecule.

[0038] As used herein, the term "proliferative disease" refers to a disease caused by abnormal expansion of cells due to enlargement. Proliferative diseases can be associated with: 1) pathological proliferation of normally quiescent cells; 2) pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenase, gelatinase, and elastase); 4) pathological angiogenesis, such as proliferative retinopathy and tumor metastasis; or 5) escape from host immune surveillance and elimination of neoplastic cells.

[0039] Such proliferative diseases include cancer (ie, malignant neoplasms), benign neoplasms, and angiogenesis.

[0040] In one embodiment, the cancer can be a blood cancer or a solid cancer.

[0041] The blood cancer may be selected from the group consisting of, but not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma and lymphoma.

[0042] The solid cancer may be selected from the group consisting of, but not limited to, breast cancer, colorectal cancer, head and neck cancer, lung cancer, gastric cancer, brain cancer, skin cancer, colon cancer, prostate cancer, bladder cancer, kidney cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, rectal cancer, anal cancer, urethral cancer, ovarian cancer, esophageal cancer, and pancreatic cancer. In addition, the cancer may be selected from any one or more of the group consisting of gastric cancer, breast cancer, lung cancer, liver cancer, esophageal cancer, and prostate cancer that are resistant to anticancer drugs (e.g., multidrug resistance). Furthermore, the cancer may be a metastatic cancer, i.e., cancer cells separated from the initial site of occurrence spread and proliferate to other sites through the blood, lymphatic vessels, etc.

[0043] In one embodiment, the benign neoplasms may include adenomas, fibromas, hemangiomas, tuberous sclerosis, and lipomas.

[0044] As used herein, the term "therapeutic agent" or "pharmaceutical composition" refers to a molecule or compound that imparts some beneficial effect when administered to a subject. Beneficial effects include: enabling diagnostic decisions; ameliorating a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally managing a disease, symptom, disorder, or condition.

[0045] In this specification, the terms "treat" or "therapeutic" or "mitigated" or "improved" are used interchangeably. These terms refer to methods of obtaining advantageous or desired results, including but not limited to therapeutic benefit and / or preventive benefit. Therapeutic benefit refers to any therapeutically significant improvement in one or more diseases, conditions or symptoms being treated or the effect it produces. In preventive benefit, the composition can be administered to a subject at risk of developing a particular disease, condition or symptom, or to a subject reporting one or more physiological symptoms of a disease even if the disease, condition or condition is not present.

[0046] In this specification, the term "effective amount" or "therapeutically effective amount" refers to the amount of an agent sufficient to cause a favorable or desired result. The therapeutically effective amount can vary according to one or more of the subject and condition being treated, the subject's weight and age, the severity of the condition, the mode of administration, etc., which can be easily determined by a person skilled in the art. The term is also applicable to the dose of providing an image to be detected by any of the imaging methods described herein. The specific dose can vary according to the specific agent selected, the drug administration therapy followed, whether it is administered in combination with other compounds, the time of administration, the tissue imaged, and the body delivery system loaded therein.

[0047] The pharmaceutical composition can be administered parenterally during clinical administration and can be used in the form of conventional pharmaceutical preparations. Parenteral administration can refer to administration by administration routes other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal (Nasal), inhalation, ocular and subcutaneous. When the pharmaceutical composition of the present invention is used as a medicine, it can further contain one or more active ingredients that exhibit the same or similar functions.

[0048] When the pharmaceutical composition is formulated, a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant and surfactant is used for preparation. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can use vegetable oils such as propylene glycol, polyethylene glycol, and olive oil; injectable esters such as ethyl oleate, etc. The base of the suppository can use Witepsol, polyethylene glycol (Macrogol), Tween (Tween) 61, cocoa butter (cacao butter), laurinum, and glycerin gelatin, etc.

[0049] Furthermore, the pharmaceutical composition can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and in order to improve stability or absorbability, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as agents.

[0050] The effective dosage of the pharmaceutical composition is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and can be administered 1 to 3 times a day.

[0051] In this specification, the terms "subject", "individual" and "patient" are used interchangeably and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, murines, monkeys, humans, farm animals, sports animals, and pets. They also include tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0052] On the other hand, a protein corona shield nanoparticle (PCSN) is provided, which comprises the following composition:

[0053] a) Nanoparticles capable of loading drugs; b) Fusion protein containing glutathione-S-transferase (GST) bound to the surface of nanoparticles.

[0054] In addition, the present invention provides a nanoparticle drug carrier or drug complex with protein corona shielding, wherein the nanoparticle is loaded with drugs.

[0055] In addition, the present invention provides a method for preparing a nanoparticle drug carrier or drug complex with protein corona shielding, comprising the following steps i) to iii):

[0056] i) binding a linker (e.g., GSH) to the surface of the nanoparticles; ii) loading a drug into the interior or surface of the nanoparticles bound to the linker in step i); and iii) forming a protein corona shield (PCS) by coating the surface of the nanoparticles loaded with the drug in step ii) with GST fusion protein.

[0057] The nanoparticles with the protein corona shield first produce a protein corona shield with the fusion protein pre-coated on the surface, so that a corona surrounded by serum proteins will not be formed in the in vivo environment, thereby being able to escape the immune response caused by macrophages, and having a significant stealth effect. Therefore, the nanoparticles with the protein corona shield (PCSN) can not only maintain a residual time in the body, but also improve the targeting ability to target cells, and can be effectively delivered to target cells, so they can be effectively used as targeted therapeutic agents.

[0058] Effects of the Invention

[0059] According to one aspect, the fusion protein and the drug carrier or drug complex comprising the fusion protein can not only maintain a sustained residual time in the body, but also improve the targeting ability to target cells and can be effectively delivered to target cells, thus having the function of being an effective targeted therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The figure shows the results of SDS-PAGE analysis of GST-HER2 Afb.

[0061] Figure 2 The images show the HER2 expression levels in the HER2-positive breast cancer cell line SK-BR3, the gastric cancer cell line NCI-N87, and the HER2-negative breast cancer cell line MDA-MB231.

[0062] Figure 3 It is a curve graph showing the cell survival rate of HER2-positive breast cancer cell line SK-BR3, gastric cancer cell lines NCI-N87, MKN45, and HER2-negative breast cancer cell line MDA-MB231 after treatment with KMD111.

[0063] Figure 4 The graph shows the results of changes in body weight according to the dose of KMD111 in a mouse animal model transplanted with the gastric cancer cell line MKN45.

[0064] Figure 5 The graph shows the results of confirming the anti-tumor effect of KMD111 according to the dose in a mouse animal model transplanted with the gastric cancer cell line MKN45. DETAILED DESCRIPTION

[0065] Preferred embodiments are given below to help understand the present invention. However, the embodiments provided below are only for easier understanding of the present invention, rather than limiting the content of the present invention. The embodiments can be variously changed, and the embodiments are not limited to the embodiments disclosed below, and can also be implemented in various forms.

[0066] Example 1 Preparation of Affibody and Glutathione-S-transferase (GST) Fusion Protein

[0067] In the present invention, a protein corona shield is formed on the surface of nanoparticles used as drug carriers, thereby preparing drug carriers with improved stability and targeting ability in the in vivo environment. To this end, a fusion protein capable of constructing a protein corona shield is first prepared. The fusion protein is expressed in the form of an affinity body (Afb) that can specifically bind to a receptor on the surface of cancer cells and combined with GST.

[0068] Specifically, in the present invention, HER2 Afb that specifically binds to HER2 is used as Afb. GST protein is introduced into human GSTA1 (P08263) of SEQ ID NO: 13, and after the linker sequence SGGGSGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 1) is bound to the C-terminal end of GST, HER2-affine (ZHER2: 342) of SEQ ID NO: 14 is introduced. Based on the amino acid sequence of the HER2-affine-GST fusion protein of SEQ ID NO: 15, codon optimization is performed to induce expression in Escherichia coli, and then the encoding gene (SEQ ID NO: 16) is synthesized by Invitrogen GeneArt gene synthesis (ThermoFisher) and inserted into the protein expression plasmid pET151 / D-TOPO.

[0069] The plasmid constructed above was inserted (transformation) into the E. coli BL21 (DE3) strain and cultured. The expression of the fusion protein was induced by treating the culture medium with IPTG, and the cells were cultured at 30°C for 16 hours and centrifuged. The obtained cultured cells were suspended in 50mM PBS and then crushed using a high-pressure cell crusher. After crushing, centrifugation was performed to separate and recover the supernatant containing the fusion protein, and then HER2-affine-GST was purified by NI-NTA affinity chromatography using an FPLC device. The purified protein was analyzed for protein purity and molecular weight by SDS-PAGE.

[0070] Figure 1 The figure shows the results of SDS-PAGE analysis of HER2-affine-GST.

[0071] Table 1 shows the results of quantitative quantification of protein purity by SDS-PAGE analysis of HER2-Affibody-GST.

[0072] [Table 1]

[0073]

[0074] like Figure 1 As shown in Table 1, HER2-Affibody-GST protein was purified with a purity of 99.5%.

[0075] Example 2 Preparation of drug carrier with protein corona shield (PCS)

[0076] <2-1> Preparation of mesoporous silica nanoparticles

[0077] As the basic structure of drug carrier, mesoporous silica nanoparticles (MSN) were prepared.

[0078] First, in order to prepare MSNs with a diameter of less than 50nm, 1.53g of CTAB and 0.3g of tetraethylammonium hydroxide (TEAH) solution were added to 100g of deionized water, and then stirred at 80°C for 1 hour to dissolve. After CTAB and TEAH were completely dissolved, 14.45g of TEOS was added and further stirred at 800rpm for 2 hours. After the stirred liquid was in a white solid state, it was filtered with a vacuum filter, washed with deionized water, and dried in air at 70°C to obtain a dry product. The obtained dry product was homogenized in an agate mortar and calcined at 550°C for 5 hours to finally obtain mesoporous silica nanoparticles (MSNs).

[0079] <2-2> Preparation of Nanoparticles with Surface-bound GSH

[0080] As a preparation process of the protein corona shielded nanoparticles of the present invention, nanoparticles (GSH-modified particles, MMSN) were prepared in which glutathione (GSH) was bound to the surface through thiol-ene click chemistry.

[0081] 100 mg of MSN prepared in Example <2-1> and 1 mL of 3-(trimethoxysily)propyl acrylate were mixed in 18 mL of toluene. The mixed solution was stirred and reacted at 60°C for 24 hours. After the reaction, the reacted MSN was washed with ethanol and deionized water, and then added to 16 mL of DMF to prepare MSN. 100 mg of GSH was dissolved in 2 mL of deionized water to prepare GSH to form a shield. Then, the prepared MSN-containing solution and the GSH aqueous solution were mixed, 40 μL of pyridine was added, vortexed and stirred. After stirring, the mixed solution was placed at room temperature for 72 hours to induce the reaction. After the reaction was completed, the nanoparticles were washed with ethanol 3 times and vacuum dried at room temperature to finally obtain nanoparticles (MMSN) with GSH bound to the surface.

[0082] <2-3> Preparation of drug-loaded nanoparticles

[0083] In order to prepare protein corona shield nanoparticles (PCSN) to be used as drug carriers in the present invention, nanoparticles using GST-Afb as protein corona shield were prepared. Since MSN can be used as a carrier for loading substances through internal and surface chemical functional groups, in the present invention, MMSN was prepared by binding GSH to the surface of MSN, and forming a protein corona shield bound thereto by GST, thereby preparing PCSN.

[0084] Specifically, in order to load the drug into the GSH-MSN prepared in Example <2-2>, 5 mg of GSH-MSN was dispersed in 1 mL of DMSO, and then slowly added to 1 mL of DMSO solution (10 mg / mL) in which SN-38 was dissolved, and stirred at room temperature for 12 hours. After stirring, the GSH-SN-38 MSN loaded with SN-38 was recovered by centrifugation, washed 3 times with deionized water, and then vacuum dried. The drug loading ratio was calculated using the following formula.

[0085] [Formula 1]

[0086] Drug loading (%) = the mass of drug in GSH-MSN / the mass of GSH-MSN × 100

[0087] After that, in order to prepare the protein corona shielded nanoparticles (PCSN), 1 mg of HER2-affine-GST protein was prepared and dissolved in 2 mL of PBS for use. After 1 mg of SN-38-loaded GSH-SN-38 MSN was dispersed in 3 mL of PBS, the prepared protein solution was slowly added dropwise under stirring at 4°C to mix, and then stirred for another 2 hours. After stirring, the unbound protein was removed by centrifugation and washed 3 times with PBS to finally obtain PCSN (KMD111), i.e., HER2-affine-GST-SN-38 MSN, and stored in PBS for the following efficacy evaluation experiment.

[0088] Experimental Example 1: Confirmation of HER2 expression level in each cell

[0089] Before confirming the cytotoxicity of KMD111 prepared in Example 2, the HER2 expression level of each cell line was first confirmed. The HER2 receptor is a cell membrane receptor belonging to the ERBB / HER growth factor group (growth factor superfamily), and the HER2 gene is a well-known primary cancer gene. The KMD111 candidate substance is a targeted drug delivery system that combines an affinity body that specifically targets HER2 with nanoparticles loaded with SN-38, which is a topoisomerase series that inhibits DNA cloning. It is expected that the higher the HER2 expression level, the greater the cell apoptosis effect.

[0090] The cells used in the experiment were human breast cancer cell lines SKBR3, MDA-MB231 and human gastric cancer cell lines NCI-N87, MKN4 purchased from the Korean Cell Line Bank. The cells were cultured in RPMI-1640 (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 1% streptomycin-penicillin (Invitrogen, Carlsbad, CA, USA) in a 37°C, 5% CO2 incubator. The cells used in each experiment were subcultured when they grew to 80%-90% density.

[0091] Specifically, the HER2 expression level of the cell line used in the experiment was confirmed by Western blot. When the cell line grew to a density of about 80%-90% in the culture dish, the cells were broken with RIPA buffer supplemented with protease / phosphatase inhibitor, and then centrifuged to obtain cell lysate. After loading and developing 10ug of protein per well in Nu-PAGE 4-12% Bis-Tris gel, it was transferred to a PVDF membrane, and then the PVDF membrane of the transferred protein was blocked, and the signal was obtained by sequentially reacting the primary antibody, secondary antibody, and HRP substrate. The results are as follows: Figure 2 shown.

[0092] like Figure 2 As shown, it has been confirmed that the HER2 expression level in the HER2-negative breast cancer cell line MDA-MB231 is very low, while the HER2-positive breast cancer cell line SKBR3 and the gastric cancer cell line NCI-N87 express a large amount of HER2.

[0093] Experimental Example 2 Confirmation of cytotoxicity of HER2-affibroid-GST-SN-38 (KMD111) on target cells

[0094] In order to confirm the drug delivery ability of KMD111 prepared in Example 2 to target cells, the cytotoxicity to target cells was confirmed.

[0095] Specifically, the HER2-negative human breast cancer cell line MDA-MB231 cells, the breast cancer cell line SK-BR3 cells overexpressing the receptor recognized by HER2 Afb, and the gastric cancer cell lines NCI-N87 and MKN-45 were used as the normal control group at a concentration of 1×10 4 After cells / well were dispensed into 96-well plates and cultured for 24 hours, they were treated with different concentrations of KMD111 and cultured for 72 hours. Using the concentration of SN-38 as a standard, each cell was treated with concentrations of 3.1, 6.2, 12.5, 25, 50, 100, and 200 ng / mL. In the culture plate, 96 wells were treated with cck-8 (Dojin Chemical Research Institute, Japan) at 10 μL per well and cultured for 1 hour. At this time, the highly water-soluble tetrazolium salt WST-8 is reduced by the dehydrogenase activity of the cells to produce a yellow formazan dye that is soluble in tissue culture medium, so the absorbance measurement value is proportional to the number of live cells. The absorbance at 450 nm was measured using an enzyme reader (Microplate reader, full wavelength enzyme reader microspectrophotometer, Thermo Fisher Scientific, USA), and the cell survival rate compared with the control group was displayed. The results are shown as follows. Figure 3 shown.

[0096] like Figure 3 As shown, KMD111 showed a cytotoxicity of less than 30% on MDA-MB231 cells that do not express HER2, and confirmed an apoptotic efficiency of more than 80% in SKBR3 cells, a breast cancer cell line that highly expresses HER2, and 70% in NCI-N87, a gastric cancer cell line. This confirmed that it can exert a specific apoptotic effect on HER2-positive cancer cells and can show a significant anti-cancer effect when used as a drug carrier.

[0097] Experimental Example 3 In vivo efficacy evaluation using HER2-affine-GST-SN-38 MSN (KMD111)

[0098] To confirm the anticancer effect of KMD111 on target tumor cells, the mouse xenograft model transplanted with the human gastric cancer MKN45 cell line was used to test it in an in vivo environment.

[0099] Specifically, in order to test the anticancer efficacy, 6-week-old NIG (NOD / SCID, GHBIO) mice were purchased and raised in an environment where food and water were freely available. After adapting for more than 10 days, tumor cells were transplanted. The tumor cells were HER2-positive gastric cancer MKN45 cells, and each individual was 5×10 6 The cells / 100 μL were injected subcutaneously into the specified position on the left side of the abdomen. Tumor measurements were performed 1 week later until the start of the test substance administration and the tumor size reached about 150 mm. 3 The individuals were randomly assigned to each test group and then began to receive medication.

[0100] The test group was divided into a control group PBS administration group and a test substance KMD111 administration group based on SN-38, divided into 3 mg / kg, 6 mg / kg, and 12 mg / kg administration groups, with 3 mice in each group, and intravenous injection (IV) was performed once every 3 days for a total of 8 times for 21 days, and the tumor size and weight were measured from the 11th day after cancer cell transplantation (0 day) to the end day (32 day). The tumor size, relative tumor volume (RTV), and tumor growth inhibition rate (TGI %) were calculated by measuring the long and short axis lengths of the tumor and using the following formula.

[0101] [Formula 2]

[0102] Tumor size (mm 3 ) = (length × width 2 ) / 2, (L: major axis, W: minor axis)

[0103] [Formula 3]

[0104] RTV = (tumor size on the last day) / (tumor size on the first day)

[0105] [Formula 4]

[0106] TGI %=[1-(RTV of treatment group) / (RTV of control group)]×100 (%)

[0107] The weight examination results of each experimental animal are as follows Figure 4 The analysis results of tumor size are shown in Figure 5 shown.

[0108] like Figure 4 As shown, the body weight of each group did not increase with age. Although a decrease in body weight was observed in the KMD111-administered group, no special clinical symptoms were observed and the group was in good condition.

[0109] like Figure 5 As shown, when analyzing the tumor size, the tumor size in the KMD111-administered group was significantly reduced in a concentration-dependent manner compared to the PBS-administered group. Compared with the control PBS-administered group, the tumor growth inhibition rates (TGI%) of the KMD111-3mg / kg, KMD111-6mg / kg, and KMD111-12mg / kg-administered groups were 79.5%, 83.5%, and 92.5%, respectively, confirming that tumor progression was inhibited. This means that in the xenograft mouse model transplanted with the MKN45 cell line, KMD111 inhibited tumor growth at all concentrations, and KMD111 had a significant anticancer effect on MKN45 tumors.

[0110] The above description of the present invention is only for example, and those skilled in the art will appreciate that it can be easily modified into other specific forms without changing the technical ideas or essential features of the present invention. Therefore, the embodiments described above should be understood in all aspects as exemplary rather than restrictive.

Claims

1. A drug complex comprising: Glutathione-S-transferase molecule; An antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2; a linker connecting the glutathione-S-transferase and the antibody, affibody or diabody; as well as The glutathione-S-transferase and the 7-ethyl-10-hydroxycamptothecin molecule bound to the glutathione molecule.

2. The drug complex according to claim 1, wherein The 7-ethyl-10-hydroxycamptothecin molecule is represented by the following chemical formula 1. [Chemical formula 1] 3. The drug complex according to claim 1, wherein The affibody is a HER2-specific affibody of SEQ ID NO:

14.

4. The drug complex according to claim 1, wherein The linker does not bind proteases.

5. The drug complex according to claim 1, wherein The 7-ethyl-10-hydroxycamptothecin molecules are loaded into the nanoparticles.

6. The drug complex according to claim 5, wherein The nanoparticles are any one selected from mesoporous silica nanoparticles, gold nanoparticles, magnetic nanoparticles, nucleic acid-metal organic framework nanoparticles and polymer nanoparticles.

7. A pharmaceutical composition for preventing or treating a proliferative disease, comprising a drug complex as an active ingredient, wherein the drug complex comprises: Glutathione-S-transferase molecule; An antibody, an affibody or a diabody molecule having the ability to bind to human epidermal growth factor receptor 2; a linker connecting the glutathione-S-transferase and the antibody, affibody or diabody; as well as The glutathione-S-transferase and the 7-ethyl-10-hydroxycamptothecin molecule bound to the glutathione molecule.

8. The pharmaceutical composition according to claim 7, wherein The 7-ethyl-10-hydroxycamptothecin molecule is represented by the following chemical formula 1. [Chemical formula 1] 9. The pharmaceutical composition according to claim 7, wherein The proliferative disease is any one selected from cancer, benign neoplasms and angiogenesis.

10. The pharmaceutical composition according to claim 9, wherein The cancer is any one selected from breast cancer, colorectal cancer, head and neck cancer, lung cancer, stomach cancer, brain cancer, skin cancer, colon cancer, prostate cancer, bladder cancer, kidney cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, rectal cancer, anal cancer, urethral cancer, ovarian cancer, esophageal cancer and pancreatic cancer.

11. The pharmaceutical composition according to claim 7, wherein The 7-ethyl-10-hydroxycamptothecin is loaded into the nanoparticles.

12. The pharmaceutical composition according to claim 11, wherein The nanoparticles are any one selected from mesoporous silica nanoparticles, gold nanoparticles, magnetic nanoparticles, nucleic acid-metal organic framework nanoparticles and polymer nanoparticles.

13. A method for preventing or treating a proliferative disease, the method comprising the following steps: An effective amount of the complex of claim 1 is administered to an individual in need thereof.

14. Use of the complex according to claim 1 in the preparation of a pharmaceutical preparation for preventing or treating a proliferative disease.

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