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

Abstract

The present invention relates to a fusion protein comprising glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein, and its use as a drug conjugate and a pharmaceutical composition. According to the fusion protein and the drug conjugate containing the same according to one aspect, not only can the in vivo residence time be prolonged, but also the targeting ability to target cells is improved and it can be effectively delivered to the target cells, so that it has an effect of being usefully used as a targeted therapeutic agent.

Description

Technical Field

[0001] It relates to a fusion protein comprising glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein, and its use as a drug delivery body, a drug complex, and a pharmaceutical composition.

Background Art

[0002] Nanoparticles have excellent biodistribution ability, can regulate the degree of drug release, and are used as useful tools in fields such as imaging devices and targeted therapeutic agents. Typically, when nanoparticles have a diameter of 200 nm, they may flow out near the blood vessels around the tumor, and since lymphatic vessels are not formed around the tumor and the pressure is low, it is known that the outflowed nanoparticles can continue to remain in the tumor tissue. This process is called the EPR effect (enhanced permeability and retention effect), by which the permeability and retention of the drug delivery body can be improved.

[0003] Representative examples of currently commercially available nanoparticles are Abraxane and Doxil. However, their vascular permeability varies from tumor to tumor, and when nanoparticles are administered intravenously, there may be a limitation that they rapidly accumulate in reticuloendothelial organs such as the liver and spleen because the mononuclear phagocyte system (MPS) removes them. As a result, the targeted therapeutic agent to which the nanoparticles are applied may have a reduced therapeutic effect and may show side effects due to the toxicity of the nanoparticles.

[0004] To overcome such drawbacks, a method of polyethylene glycolylation (PEGylation) to surround nanoparticles has been developed. When such a method is used, there is an advantage that the time during which nanoparticles can circulate in the in vivo circulatory system becomes longer. Conversely, the possibility of flowing into target cells decreases, and cells other than target cells may be targeted through non-specific binding, which may also reduce the therapeutic efficiency.

[0005] When using nanoparticles as drug delivery carriers, a strategy has been proposed to induce structural changes in the nanoparticles in order to promote the EPR effect along with the targeting ability for drug delivery. Specifically, attempts have continued to improve the targeting ability of the drug delivery carrier by inducing structural changes such as coating the surface of the nanoparticles with an antibody, protein, or peptide that can bind to receptors overexpressed in cancer cells. However, even with these methods, the tumor targeting efficiency has not effectively increased. Instead, it provides a target ligand that can more rapidly remove nanoparticles through the in vivo immune response via MPS, and there is a limitation in that it does not show a significant therapeutic effect in terms of the overall tumor treatment effect.

[0006] Theoretically, when exposed to a physiological environment, the surface of the nanoparticles is naturally surrounded by other biomolecules in a direction that reduces the surface energy due to the combined action of the arrangement of water molecules according to entropy, charge compensation on the particle surface, and the exposure of hydrophobic portions. At this time, various biomolecules are non-specifically adsorbed on the surface of the nanoparticles, and such a form is called a protein corona. The protein corona is surrounded by other molecules, and its original molecular characteristics change, blocking the targeting ability to target cells or organs and various biological functions exhibited by the nanoparticles. Therefore, research is underway to regulate the protein corona so that nanoparticles can be applied at the clinical level as a targeted therapeutic agent.

[0007] Therefore, in the process of manufacturing nanoparticle-based targeted therapeutics, a method has been developed to first form and then regulate a protein corona on the surface of the nanoparticles. For this purpose, in order to avoid blocking the targeting ability by the protein corona, methods are known to minimize the interaction with proteins in serum by changing the surface of the nanoparticles to zwitterionic, PEG, carbohydrate residues, etc. In addition, the nanoparticles can be pre-coated with dysopsonic proteins to regulate the protein corona through the circulation of the desired proteins in plasma. As a result, the nanoparticles pre-coated with the protein corona have the effect of increasing the colloidal stability and being able to sustain the circulation time in the blood without being removed by the MPS. However, such methods may also limit the targeting ability to the target, and there are limitations in clinical application in that the biochemical effects due to the biological interaction between the protein used for the pre-coating and the nanoparticles are not known.

Summary of the Invention

Problems to be Solved by the Invention

[0008] One aspect provides a drug conjugate comprising a glutathione-S-transferase (GST) molecule, an antibody having binding ability to HER2 (human epidermal growth factor receptor2), an affibody or a diabody molecule, a linker linking the glutathione-S-transferase and the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GSH (Glutathione) molecule.

[0009] Another aspect is to provide a pharmaceutical composition for preventing or treating a proliferative disease, which comprises a glutathione-S-transferase (GST) molecule, an antibody having the ability to bind to HER2 (human epidermal growth factor receptor 2), an affibody or a diabody molecule, a linker that links the glutathione-S-transferase and the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GST (Glutathione) molecule.

[0010] Another aspect is to provide a method for preventing or treating a proliferative disease, which comprises administering to an individual in need thereof, in an effective amount, a drug conjugate comprising a glutathione-S-transferase (GST) molecule, an antibody having the ability to bind to HER2 (human epidermal growth factor receptor 2), an affibody or a diabody molecule, a linker that links the glutathione-S-transferase and the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GSH (Glutathione) molecule.

[0011] Another aspect is to provide the use of a drug conjugate comprising a glutathione-S-transferase (GST) molecule, an antibody having the ability to bind to HER2 (human epidermal growth factor receptor 2), an affibody or a diabody molecule for the manufacture of a pharmaceutical preparation for the prevention or treatment of a proliferative disease, a linker that links the glutathione-S-transferase and the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GSH (Glutathione) molecule. Means for solving the invention

[0012] One aspect is to provide a drug conjugate comprising a glutathione-S-transferase (GST) molecule, an antibody having the ability to bind to HER2 (human epidermal growth factor receptor 2), an affibody or a diabody molecule, a linker that links the glutathione-S-transferase and the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GSH (Glutathione) molecule.

[0013] As used herein, the term "antibody" means a specific protein molecule directed against an antigenic site as a term known in the art. For the purposes of this specification, an antibody means an antibody that specifically binds to a target cell or a receptor expressed on the target cell, and such an antibody can be obtained by cloning each gene into an expression vector according to a conventional method, obtaining the protein encoded by the marker gene, and producing it from the obtained protein by a conventional method. This includes partial peptides made from the protein, and the partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody in this specification is not particularly limited, and polyclonal antibodies, monoclonal antibodies, humanized antibodies, or antigen-binding fragments that are part of those having antigen-binding properties are also included in the antibodies of this specification, and all immunoglobulin antibodies can be included.

[0014] As used herein, the term "affibody molecule" may mean an antibody mimic that can bind to a specific target protein (receptor). Generally, an affibody molecule may be composed of 20 to 150 amino acid residues and may consist of 2 to 10 alpha helices. More specifically, 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. Furthermore, without being limited thereto, it includes all affibody molecules that can recognize a specific receptor or target protein of a cell. Examples of the target receptor or target protein that the affibody molecule can recognize are amyloid β peptide, synuclein (e.g., α-synuclein), apolipoprotein (e.g., apolipoprotein A1), complement factor (e.g., C5), carbonic anhydrase (e.g., CAIX), interleukin-2 receptor α 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, interleukin (e.g., IL-1, IL-6, IL-8, IL-17), insulin, staphylococcal protein A domain, Raf-1, LOV domain (Light-oxygen-voltage-sensing domain), or RSV G protein. Information regarding 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, and the entire content of the said literature is incorporated herein by reference.

[0015] As used herein, the term "linker" means a molecule that links 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 contain Gly, Asn, and Ser residues, and can also contain neutral amino acids such as Thr and Ala. Amino acid sequences suitable for peptide linkers are known in the art. Furthermore, the copy number "n" can be adjusted in consideration of achieving an appropriate separation between functional moieties or optimizing the linker to maintain essential inter-moiety interactions.

[0016] In one embodiment, the peptide linker can be a protease-resistant linker. Being protease-resistant specifically means that it does not bind to, and / or is not cleaved by, a protease, and / or remains stable and / or maintains its activity when in contact with a protease.

[0017] In one embodiment, the peptide linker can be a flexible linker containing G, S, and / or T residues. Other flexible linkers are known in the art, and there can be, for example, G and S linkers to which polar amino acid residues are added not only to improve water solubility but also amino acid residues such as T and A are added to maintain flexibility. Specifically, the linker can have a general formula selected from (GpSs)n and (SpGs)n, where, independently, p is an integer from 1 to 10, s is 0 or an integer from 0 to 10, p + s is an integer of 20 or less, 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), where the n is an integer from 1 to 20, or from 1 to 10.

[0018] Another aspect provides a polynucleotide encoding a fusion protein to which an antibody, an affibody, or a bispecific antibody molecule capable of binding to the GST molecule and HER2 is bound.

[0019] As used herein, the term "polynucleotide" means a polymer of deoxyribonucleotides or ribonucleotides that exists in single-stranded or double-stranded form. It includes RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes not only natural polynucleotides but also analogues thereof in which the sugar or base moieties are modified, unless otherwise specified. In one embodiment, the polynucleotide is a short-chain polynucleotide.

[0020] Another aspect provides a vector containing the polynucleotide.

[0021] As used herein, the term "vector" refers to a gene construct that can express a target protein in a suitable host cell and contains regulatory elements operably linked so that the gene insert is expressed. A vector according to one embodiment may contain 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. Further, the vector may be an expression vector capable of stably expressing the fusion protein in a host cell. As the expression vector, those commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms can be used. The recombinant vector can be constructed by various methods known in the art. For example, the vector may contain a selectable marker for selecting a host cell containing the vector, and may contain an origin of replication if it is a replicable vector. Furthermore, the vector can be self-replicating or introduced into the host DNA, and the vector may be selected from the group consisting of plasmids, lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, herpes simplex viruses, and vaccinia viruses.

[0022] The vector includes a promoter that is operable in animal cells, such as mammalian cells. Suitable promoters according to one embodiment include promoters derived from mammalian viruses and promoters derived from the genomes of mammalian cells, such as the CMV (Cytomegalovirus) promoter, the U6 promoter, the H1 promoter, the MLV (Murine Leukemia Virus) LTR (Long terminal repeat) promoter, the adenovirus early promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the HSV tk promoter, the RSV promoter, the EF1α promoter, the metallothionein promoter, the β-actin promoter, the promoter of the human IL-2 gene, the promoter of the human IFN gene, the promoter of the human IL-4 gene, the promoter of the human lymphotoxin gene, the promoter of the human GM-CSF gene, the human phosphoglycerate kinase (PGK) promoter, the mouse phosphoglycerate kinase (PGK) promoter, and the Survivin promoter.

[0023] Furthermore, in the vector, the above-described fusion protein may be operably linked to a promoter. As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription regulatory factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequence.

[0024] Another aspect provides a host cell comprising the fusion protein, polynucleotide, or vector.

[0025] The cells, for example, eukaryotic cells, can be cells of yeast, mold, protozoa, plants, higher plants and insects, or amphibians, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), graft cells, and primary cell cultures (in vitro and ex vivo) commonly used in the art, and in vivo cells, and / or mammalian cells including humans. Further, the organism can be yeast, mold, protozoa, plants, higher plants and insects, amphibians, or mammals. Further, the cells can be animal cells or plant cells.

[0026] The types of pharmaceutically active ingredients that can be delivered to an individual using a drug delivery body include anticancer agents, contrast agents (dyes), hormonal agents, antihormonal agents, vitamin agents, calcium agents, inorganic preparations, saccharide agents, organic acid preparations, protein amino acid preparations, antidotes, enzyme preparations, metabolic preparations, diabetes combination agents, tissue regenerating agents, chlorophyll preparations, pigment preparations, tumor agents, tumor therapeutic agents, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapeutic agents, antibiotic preparations, antiviral agents, combined antibiotic preparations, chemotherapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospira sera, blood preparations, biological agents, analgesics, immunogenic molecules, antihistamines, allergy medications, non-specific immunogen preparations, anesthetics, stimulants, psychoneural agents, low molecular weight compounds, nucleic acids, aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNA, and microRNA, etc.

[0027] SN-38 can be an anticancer agent and can include its pharmaceutically acceptable salts. "SN-38 (7-ethyl-10-hydroxycamptothecin)" is the active ingredient of irinotecan (also known as "CPT-11") and exhibits antitumor effects by inhibiting type I DNA topoisomerase activity. It can exhibit cytotoxic activity more than 1,000 times stronger against various cancer cells in vitro than irinotecan.

[0028] In the present invention, the binding of glutathione-S-transferase to SN-38 (7-ethyl-10-hydroxycamptothecin) may be due to GSH (Glutathione). That is, the SN-38 may be SN-38 to which GSH is bound. GSH functions as a binding site for glutathione-S-transferase and can link SN-38 and glutathione-S-transferase.

[0029] The SN-38 molecule may be nanoparticles carrying SN-38 or capable of carrying SN-38. The nanoparticles can be applied without limitation as long as they are nanoparticles applicable as drug delivery carriers according to the prior art. Specifically, it may be any selected from the group consisting of mesoporous silica nanoparticles (MSN), gold nanoparticles, magnetic nanoparticles, nucleic acid-metal organic framework nanoparticles, and polymer nanoparticles. Further, the nanoparticles may be those to which GSH (Glutathione) is bound. Thereby, the nanoparticles can bind to a fusion protein containing GST.

[0030] In one embodiment, the SN-38 molecule is represented by the following formula 1. [Chemical formula]

[0031] Another aspect is to provide a pharmaceutical composition for preventing or treating a proliferative disease, which comprises a glutathione-S-transferase (GST) molecule, an antibody having the ability to bind to HER2 (human epidermal growth factor receptor 2), an affibody or a diabody molecule, a linker for linking the glutathione-S-transferase and the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GST (Glutathione) molecule.

[0032] Another aspect is to provide a method for preventing or treating a proliferative disease, which comprises the step of administering, in an effective amount, to an individual in need thereof, a drug conjugate comprising a glutathione-S-transferase (GST) molecule, an antibody having the ability to bind to HER2 (human epidermal growth factor receptor 2), an affibody or a diabody molecule, a linker for linking the glutathione-S-transferase and the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GSH (Glutathione) molecule.

[0033] Another aspect provides for the use of a drug conjugate comprising a glutathione-S-transferase (GST) molecule, an antibody, affibody or diabody molecule having the ability to bind to HER2 (human epidermal growth factor receptor 2), a linker linking the glutathione-S-transferase to the antibody, affibody or diabody, and 7-ethyl-10-hydroxycamptothecin bound by the glutathione-S-transferase and a GSH (Glutathione) molecule, for the manufacture of a pharmaceutical preparation for the prevention or treatment of a proliferative disease.

[0034] As used herein, the term "proliferative disease" refers to a disease caused by abnormal expansion due to cell growth. Proliferative diseases can be associated with: 1) pathological growth of normal quiescent cells, 2) pathological movement 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) avoidance of host immune surveillance and neoplastic cell removal.

[0035] The proliferative diseases include cancer (i.e., malignant neoplasm), benign neoplasm, and angiogenesis.

[0036] In one embodiment, the cancer can be a blood cancer or a solid cancer.

[0037] The blood cancer can be selected from the group consisting of, but not limited to, Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, Chronic Myelogenous Leukemia, Multiple Myeloma and Lymphoma.

[0038] 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. Further, the cancer may be any one or more selected from the group consisting of gastric cancer, breast cancer, lung cancer, liver cancer, esophageal cancer, and prostate cancer having resistance (e.g., multi-drug resistance) to anti-cancer agents. Also, the cancer may be metastatic cancer in which cancer cells separated from the initially-occurring site metastasize and proliferate to other sites via blood, lymphatic vessels, etc.

[0039] In one embodiment, the benign neoplasm may include adenoma, fibroma, hemangioma, tuberous sclerosis, and lipoma.

[0040] As used herein, the terms "therapeutic agent" or "pharmaceutical composition" refer to a molecule or compound that provides some beneficial effect upon administration to a subject. Beneficial effects include enabling diagnostic determinations, improvement of a disease, symptom, disorder or condition, reduction or prevention of the onset of a disease, symptom, disorder or illness, and generally, addressing a disease, symptom, disorder or condition.

[0041] As used herein, the terms "treat" or "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to a method of obtaining beneficial or desired results including, but not limited to, therapeutic and / or prophylactic benefits. Therapeutic benefit means any therapeutically significant improvement or effect of one or more diseases, illnesses or symptoms under treatment. In prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, illness or symptom, or to a subject reporting one or more physiological symptoms of a disease even when the disease, illness or symptom has not yet manifested.

[0042] As used herein, the terms "effective amount" or "therapeutically effective amount" refer to an amount of an agent sufficient to produce a beneficial or desired result. The therapeutically effective amount can vary depending on 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., and can be readily determined by one of ordinary skill in the art. Further, the terms apply to the capacity to provide an image for detection by any of the imaging methods described herein. A particular dosage can vary depending on one or more of the particular agent selected, the subsequent therapy of administration, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the delivery system of the body carrying it.

[0043] The pharmaceutical composition can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations. Parenteral administration can mean administration via a route other than oral administration such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, intraocular, and subcutaneous. When using the pharmaceutical composition of the present invention as a pharmaceutical, it can further contain one or more active ingredients showing the same or similar functions.

[0044] When formulating the pharmaceutical composition, it is usually prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injection esters such as ethyl oleate can be used. As the base of suppositories, Witepsol, macrogol, Tween 61, cocoa butter, laurin fat, glycerogelatin, etc. can be used.

[0045] Furthermore, the pharmaceutical composition can be used in combination with several carriers acceptable as drugs, such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as drugs to enhance stability or absorbability.

[0046] The effective dose of the pharmaceutical composition is 0.01 - 100 mg / kg, preferably 0.1 - 10 mg / kg, and it can be administered once to three times a day.

[0047] As used herein, the terms "subject", "individual" and "patient" are used interchangeably herein to refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, murine, ape, human, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0048] Another aspect provides a protein corona shield nanoparticle (PCSN) having the following configuration.

[0049] a) Nanoparticles capable of carrying drugs, b) A fusion protein containing glutathione - S - transferase (GST) bound to the surface of the nanoparticles.

[0050] The present invention also provides a nanoparticle drug delivery body or drug complex having a protein corona outer layer with a drug carried inside the nanoparticles.

[0051] Furthermore, the present invention provides a method for producing a nanoparticle drug delivery system or a drug complex having a protein corona outer layer, which includes the following steps i) to iii).

[0052] i) a step of binding a linker (e.g., GSH) to the surface of the nanoparticle; ii) a step of loading a drug inside or on the surface of the nanoparticle bound to the linker in step i); iii) a step of coating the surface of the nanoparticle loaded with the drug in step ii) with a GST fusion protein to form a protein corona outer layer (PCS).

[0053] The nanoparticle having the protein corona outer layer (protein corona shield) generates a protein corona outer layer whose surface is pre-coated with a fusion protein first. Therefore, a corona layer surrounded by serum proteins is not formed in the in vivo environment, and the immune reaction of macrophages can be avoided, and a significant stealth effect can be achieved. Therefore, the nanoparticle (PCSN) having the protein corona outer layer can not only prolong the residence time in the living body, but also improve the targeting ability to target cells and can be effectively delivered to the target cells, so it can be usefully used as a target therapeutic agent.

Advantages of the Invention

[0054] According to the fusion protein and the drug delivery system or drug complex containing the same according to one aspect, not only can the residence time in the living body be prolonged, but also the targeting ability to target cells is improved, and it can be effectively delivered to the target cells. Therefore, it can be usefully used as a target therapeutic agent.

Brief Description of the Drawings

[0055]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0056] Hereinafter, preferred embodiments are presented to assist in understanding the present invention. However, the following embodiments are provided to more easily understand the present invention, and the content of the present invention is not limited by the following embodiments. The embodiments can be subjected to various conversions, but the embodiments are not limited to the embodiments disclosed below and can be implemented in various forms.

[0057] Embodiment 1. Preparation of Affibody and Glutathione-S-Transferase (GST) Fusion Protein

[0058] In the present invention, an outer layer of a protein corona was formed on the surface of nanoparticles for use as a drug delivery carrier to produce a drug delivery carrier with improved stability and targeting ability even in the in vivo environment. For this purpose, first, a fusion protein that can constitute the outer layer of the protein corona was prepared. The fusion protein was expressed in a form in which an affibody (Afb) that can specifically bind to a receptor on the cancer cell surface and GST are bound.

[0059] Specifically, in the present invention, HER2 Afb that specifically binds to HER2 was used as Afb. The GST protein was introduced with human GSTA1 (P08263) of SEQ ID NO: 13, and after binding the linker sequence SGGGSGGGSGGSGGGSGGGSGG (SEQ ID NO: 1) to the C-terminal end of GST, the HER2-affibody (ZHER2:342) of SEQ ID NO: 14 was introduced. After performing codon optimization for inducing expression in E. coli based on the amino acid sequence of the HER2-affibody-GST fusion protein of SEQ ID NO: 15, the encrypted gene (SEQ ID NO: 16) was synthesized by Invitrogen GeneArt gene synthesis (ThermoFisher) and inserted into the protein expression plasmid pET151 / D-TOPO.

[0060] The constructed plasmid was inserted (transformed) into E. coli BL21(DE3) strain and cultured. The fusion protein was induced to express by treating the culture solution with IPTG, cultured at 30 °C for 16 hours, and the cells were obtained by centrifugation. The obtained cultured cells were suspended in 50 mM PBS, and then the cells were disrupted using a high-pressure cell disruptor. After disruption, centrifugation was performed to separate and recover the supernatant containing the fusion protein, and then HER2-affibody-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 analysis.

[0061] Figure 1 is a graph showing the SDS-PAGE analysis results of HER2-affibody-GST.

[0062] Table 1 shows the results of quantitatively numericalizing the protein purity by SDS-PAGE analysis of HER2-affibody-GST.

[0063]

Table 1

[0064] As shown in FIG. 1 and Table 1, a HER2-affibody-GST protein with a purity of 99.5% was purified.

[0065] Embodiment 2. Preparation of a drug delivery body having a protein corona outer layer (PCS)

[0066] <2-1>Preparation of porous silica nanoparticles

[0067] As the basic structure of the drug delivery body, porous silica nanoparticles (mesoporous silica nanoparticle, MSN) were prepared.

[0068] First, to prepare MSN with a diameter of 50 nm or less, 1.53 g of CTAB and 0.3 g of tetraethylammonium hydroxide solution (Tetraethylammonium hodioxide, TEAH) were added to 100 g of deionized water, and then stirred at 80° C. for 1 hour to dissolve them. After CTAB and TEAH were completely dissolved, 14.45 g of TEOS was added, and further stirred at 800 rpm for 2 hours. When the stirred solution showed a white solid phase, it was filtered with a vacuum filter, then washed with deionized water, and dried in air at 70° C. to obtain a dried product. The obtained dried product was homogenized in an agate mortar and then calcined at 550° C. for 5 hours to finally obtain porous silica nanoparticles (MSN).

[0069] <2-2>Production of nanoparticles with GSH surface-bound

[0070] As a process for producing the nanoparticles having the protein corona outer layer of the present invention, nanoparticles (GSH-modified particle, MMSN) with glutathione (GSH) bound to the surface by thiol-ene click chemistry were prepared.

[0071] 100 mg of MSN prepared in the above Embodiment <2-1> and 1 ml of 3-(trimethoxysilyl)propyl acrylate were mixed in 18 ml of toluene. The mixed solution was stirred at 60 °C for 24 hours for reaction. After the reaction, the MSN reacted with ethanol and deionized water was washed, and then added to 16 ml of DMF to prepare MSN. GSH for forming the outer layer was prepared by dissolving 100 mg of GSH in 2 ml of deionized water. Next, the prepared MSN-containing solution and the GSH aqueous solution were mixed, 40 μl of pyridine was added, and vortexed and stirred. After stirring, the mixed solution was left at room temperature for 72 hours to induce the reaction. After completion of the reaction, the nanoparticles were washed three times with ethanol and vacuum dried at room temperature to finally obtain nanoparticles (MMSN) with GSH bound to the surface.

[0072] <2-3> Production of Drug-Loaded Nanoparticles

[0073] In the present invention, in order to produce nanoparticles (protein corona shield nanoparticle, PCSN) having a protein corona outer layer to be used as a drug delivery carrier, nanoparticles applying GST-Afb as the protein corona outer layer were produced. Since MSN is used as a carrier capable of carrying substances via chemical functional groups inside and on the surface, in the present invention, GSH was bound to the MSN surface to produce MMSN, and a protein corona outer layer bound via GST was formed thereon to produce PCSN.

[0074] Specifically, in order to carry a drug on the GSH-MSN produced in the above Embodiment <2-2>, 5 mg of GSH-MSN was dispersed in 1 ml of DMSO, and then slowly added to 1 ml of a 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 carrying SN-38 was centrifuged and recovered, washed three times with DI water, and vacuum dried. The ratio of the drug carried was calculated by the following formula. [Equation 1] Drug loading capacity (%) = Amount of drug in GSH-MSN / Amount of GSH-MSN × 100

[0075] Next, to prepare nanoparticles with a protein corona outer layer (PCSN), 1 mg of HER2-affibody-GST protein was prepared and dissolved in 2 ml of PBS. After dispersing 1 mg of GSH-SN-38 MSN carrying SN-38 in 3 ml of PBS, the prepared protein solution was slowly added dropwise with stirring at 4°C and mixed, and then stirred for another 2 hours. After stirring, centrifugation was performed to remove unbound protein, and it was washed 3 times with PBS. Finally, PCSN (KMD111), which is HER2-affibody-GST-SN-38 MSN, was obtained and used in the following efficacy evaluation experiment while stored in PBS.

[0076] Experimental Example 1. Confirmation of HER2 expression level in each cell

[0077] Before confirming the cytotoxicity of KMD111 prepared in the above Embodiment 2, first, the HER2 expression level of each cell line was confirmed. The HER2 receptor is a cell membrane receptor belonging to the ERBB / HER growth factor superfamily, and the HER2 gene is a well-known primary cancer gene. The KMD111 candidate substance is a targeted drug delivery system in which an Affibody that specifically targets HER2 is bound to nanoparticles loaded with SN-38, which is a topoisomerase system that suppresses DNA replication. It was expected that the higher the HER2 expression level, the greater the apoptotic effect.

[0078] The cells used in the experiments were the human breast cancer cell lines SKBR3 and MDA-MB231, and the human gastric cancer cell lines NCI-N87 and MKN4, which were purchased from the Korean Cell Line Bank. They were cultured in RPMI-1640 medium (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) in a 37 °C, 5% CO2 incubator. The cells used in each experiment were passaged when they grew to a density of about 80% - 90%.

[0079] Specifically, the HER2 expression levels of the cell lines used in the experiment were confirmed by Western blot. When the cell lines grew to a density of about 80% - 90% in the culture dish, the cells were lysed with RIPA buffer supplemented with protease / phosphatase inhibitor, and then centrifuged to obtain cell lysate. 10 μg of protein per well was loaded onto a Nu-PAGE 4-12% Bis-Tris gel and electrophoresed, then transferred to a PVDF membrane. The PVDF membrane onto which the protein was transferred was blocked, and reacted with the primary antibody, secondary antibody, and HRP substrate in sequence to obtain a signal, and the results are shown in Figure 2.

[0080] As shown in Figure 2, it was confirmed that the HER2 expression level was very low in MDA-MB231, a HER2-negative breast cancer cell line, while SKBR3, a HER2-positive breast cancer cell line, and the gastric cancer cell line NCI-N87 highly expressed HER2.

[0081] Experimental Example 2. Confirmation of the cytotoxicity of HER2-affibody-GST-SN-38 (KMD111) against target cells

[0082] To confirm the drug delivery ability of KMD111 prepared in the above Embodiment 2 to target cells, the cytotoxicity against the target cells was confirmed.

[0083] Specifically, MDA-MB231 cells, a human breast cancer cell line that is HER2 negative and used as a normal control group, SK-BR3 cells, a breast cancer cell line that overexpresses the receptor recognized by HER2 Afb, NCI-N87, a gastric cancer cell line, and MKN-45 were placed in a 96-well plate (well plate) at 1×10 4 cells / well, cultured for 24 hours, then treated with KMD111 at different concentrations and cultured for 72 hours. Each cell was treated with SN-38 at concentration standards of 3.1, 6.2, 12.5, 25, 50, 100, 200 ng / mL. After treating 10 μL of cck-8 (Dojindo, Japan) in each well of the culture plate, it was cultured for 1 hour. At this time, the highly water-soluble tetrazolium salt WST-8 decreases due to the dehydrogenase activity of the cells to generate a yellow formazan dye that dissolves in the tissue culture medium, so the measured absorbance value is proportional to the number of viable cells. The absorbance was measured at 450 nm with a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer, Thermo fisher scientific, USA), and the cell viability was shown compared with the control group. The results are shown in Figure 3.

[0084] As shown in Figure 3, KMD111 showed cytotoxicity within 30% against MDA-MB231 cells that do not express HER2, more than 80% in SKBR3 cells, a breast cancer cell line with high HER2 expression, and an apoptosis efficacy of 70% in NCI-N87, a gastric cancer cell line. Thus, it was confirmed that KMD111 can specifically show an apoptotic effect on HER2-positive cancer cells and can show a significant anti-cancer effect when used as a drug delivery vehicle.

[0085] Experimental Example 3. In vivo efficacy evaluation using HER2-affibody-GST-SN-38 MSN (KMD111)

[0086] To confirm the anti-cancer effect of KMD111 against target tumor cells, an in vivo evaluation was performed using a mouse xenograft model transplanted with human gastric cancer MKN45 cell line.

[0087] Specifically, for the anti-cancer effect evaluation, 6-week-old NIG (NOD / SCID, Hitech Bio Inc.) mice were purchased and purified for more than 10 days while being bred in an environment where feed and drinking water could be freely provided, and then tumor cells were transplanted. The tumor cells were HER2-positive gastric cancer MKN45 cells, which were subcutaneously administered at 5×10 6 cells / 100 μL at a fixed position in the left flank. Tumor measurement was performed from 1 week after transplantation until the start of test substance administration. Individuals with a tumor size of approximately 150 mm 3 were selected and randomly assigned to each test group, and then administration was started.

[0088] The test groups were divided into a control group receiving PBS administration and test substance KMD111 administration groups at 3 mg / kg, 6 mg / kg, and 12 mg / kg based on SN-38, with 3 mice in each group. Intravenous injection (IV) was performed 8 times at intervals of 3 days for 21 days. Tumor size and body weight were measured from the 11th day after cancer cell transplantation (day 0) until the end day (32 days). Tumor size, relative tumor volume (RTV), and tumor growth inhibition rate (TGI%) were calculated using the following formulas by measuring the long and short axes of the tumor. [Equation 2] Tumor size (mm 3 ) = (long axis length × short axis length 2 ) / 2 [Equation 3] Relative tumor volume (RTV) = (tumor size at the end day) / (tumor size on the first day) [Equation 4] Tumor growth inhibition rate (TGI)% = [1 - (RTV of the test group) / (RTV of the control group)] × 100 (%)

[0089] The results of checking the body weights of each test animal are shown in Figure 4, and the results of analyzing the tumor size are shown in Figure 5.

[0090] As shown in Figure 4, there was no weight gain according to the age in all groups, and weight loss was confirmed in the KMD111-administered group, but no special clinical symptoms were shown and the condition was good.

[0091] As shown in Figure 5, when analyzing the tumor size, the tumor size decreased significantly in a concentration-dependent manner in the KMD111-administered group compared with the PBS-administered group. In the KMD111-administered group, the tumor growth inhibition rates (TGI%) of the KMD111-3 mg / kg, KMD111-6 mg / kg, and KMD111-12 mg / kg administered groups were 79.5%, 83.5%, and 92.5% respectively compared with the control group PBS-administered group, and it was confirmed that the progression of the tumor was suppressed. This means that in a xenograft mouse model transplanted with the MKN45 cell line, KMD111 inhibits tumor growth at all concentrations, and KMD111 has a clear anti-cancer effect on MKN45 tumors.

[0092] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains will understand that it can be easily deformed into other specific forms without changing the technical idea and essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims

1. A glutathione-S-transferase (GST) molecule, An antibody, affibody, or diabody molecule having the ability to bind to HER2 (human epidermal growth factor receptor 2), A linker that links the glutathione-S-transferase and the antibody, affibody, or diabody, and A drug conjugate comprising a 7-ethyl-10-hydroxycamptothecin molecule bound by the glutathione-S-transferase and a GSH (Glutathione) molecule.

2. The drug conjugate according to claim 1, wherein the 7-ethyl-10-hydroxycamptothecin molecule is represented by the following formula. 【Chemical Formula 1】

3. The drug conjugate according to claim 1, wherein the affibody is a HER2-specific affibody of SEQ ID NO:

14.

4. The drug conjugate according to claim 1, wherein the linker is not bound by a protease.

5. The drug conjugate according to claim 1, wherein the 7-ethyl-10-hydroxycamptothecin molecule is supported on nanoparticles.

6. The drug conjugate according to claim 5, wherein the nanoparticles are any selected from the group consisting of mesoporous silica nanoparticles (MSN), gold nanoparticles, magnetic nanoparticles, nucleic acid-metal organic framework nanoparticles, and polymer nanoparticles.

7. A glutathione-S-transferase (GST) molecule, An antibody, affibody or diabody molecule having the ability to bind to HER2 (human epidermal growth factor receptor 2), A linker that links the glutathione-S-transferase and the antibody, affibody or diabody, and A pharmaceutical composition for preventing or treating proliferative diseases, comprising as an active ingredient a drug conjugate containing a 7-ethyl-10-hydroxycamptothecin molecule bound by the glutathione-S-transferase and a GSH (Glutathione) molecule.

8. The pharmaceutical composition according to claim 7, wherein the 7-ethyl-10-hydroxycamptothecin molecule is represented by the following formula. [Chemical formula 2]

9. The pharmaceutical composition according to claim 7, wherein the proliferative disease is any one selected from the group consisting of cancer, benign neoplasm, and angiogenesis.

10. The pharmaceutical composition according to claim 9, wherein the cancer is any one selected from the group consisting of 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 supported on nanoparticles.

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

13. A method for preventing or treating a proliferative disease, comprising the step of administering an effective amount of the complex of claim 1 to an individual in need thereof.

14. Use of the complex of claim 1 for the manufacture of a pharmaceutical preparation for the prevention or treatment of a proliferative disease.

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