KR20120084900A - Pharmaceutical compositions for treatment of cancer comprising outer membrane vesicles as an active ingredient - Google Patents

Abstract

The present invention (a) outer membrane proteins (OMPs); (b) a cancer-targeting ligand bound to said envelope protein; And (c) outer membrane vesicles (OMVs) for treating cancer, including an anticancer agent, and a pharmaceutical composition comprising the same as an active ingredient. The anticancer drug delivery system through the outer membrane endoplasmic reticulum of the present invention can carry the anticancer agent into cells more efficiently compared to the conventional liposome-mediated methods, and requires a low production cost because it can be mass-produced very conveniently. Therefore, the composition using the outer membrane vesicles of the present invention can be applied to cancer treatment more efficiently and specifically compared to conventional methods for treating cancer (eg, liposome-mediated drug delivery system).

Description

Pharmaceutical Compositions for Treatment of Cancer Comprising Outer Membrane Vesicles as an Active Ingredient}

The present invention relates to outer membrane vesicles (OMVs) comprising cancer-targeting ligands and their use.

Cancer, tumors, tumor-related diseases and neoplastic diseases are very serious and often cause life-threatening conditions. Numerous studies have been conducted to identify therapeutic agents that are effective in the treatment of the above-mentioned diseases characterized by fast-proliferative cell growth. The therapeutic agent prolongs the survival of the patient, inhibits rapid-proliferative cell growth associated with tumorigenicity and is effective in neoplastic degeneration. In general, surgical surgery and radiation therapy are primarily performed for the treatment of cancer, and chemotherapy for certain cancers is effective in the treatment of cancer, even if survival is low.

Currently, most cancer therapies, including chemotherapy, radiotherapy and immunotherapy, function by inducing apoptosis indirectly in cancer cells. Due to the drawback of normal apoptotic mechanisms, the inability of cancer cells not to perform apoptosis is associated with increased resistance to chemotherapy, radiotherapy or immunotherapy-induced apoptosis. Due to apoptosis defects, the primary or acquired resistance of human cancers of different origin to recent treatment protocols is a major problem in cancer therapy (Lowe, et al. , Carcinogenesis , 21: 485 (2000); Nicholson, Nature , 407: 810 (2000).

In an attempt to improve more effective cancer treatments, therapies through specific targeting of monoclonal antibodies were developed in the 1970s. At least 15 monoclonal antibodies are currently approved for human use. Antibodies based on interactions with cell-specific markers may exhibit direct cytotoxicity according to a variety of mechanisms, for example, blocking growth factor receptors (Baselga, J. and Mendelsohn, J. Pharmacol. Ther. , 64: 127154 (1994)), induction of apoptosis (Trauth, BC, et al. , Science , 245: 301305 (1989)), formation of anti-idiotype antibodies (Fagerberg, J., et al. , Cancer Immunol. Immunother. , 38: 149159 (1994)) or antibody-dependent cellular cytotoxicity (ADCC; Steplewski, Z., et al. , Science , 221: 865867 (1983)) and complement-mediated Indirect effects such as complement-dependent cytotoxicity (CDC; Ballare, C., et al. , Cancer Immunol. Immunother. , 41: 1522 (1995)). On the other hand, the cytotoxicity induced by the antibody is often insufficient, and can be used together with various toxic substances (eg, radioisotopes and chemotherpeutic drugs) to increase the therapeutic efficacy. Recently, humanized monoclonal antibody trastuzumab (Herceptin), which targets human epidermal growth factor 2 (HER2 / neu) overexpressed in about 20-30% of breast cancers, is a breast cancer. It is used in therapy (WO 2006/098978 A1; WO 2009/042618 A1). However, trastuzumab as a breast cancer agent has many side effects despite its approval. For example, fever, nausea, vomiting, infusion reactions, diarrhea, infection, increased cold, headache, fatigue, shortness of breath, rash, neutrophil leukocyte reduction, anemia and myalgia are typical side effects.

Although monoclonal antibodies offer very attractive properties for tumor targeting, monoclonal antibodies not only have high cost, various side effects and limited effects, but also due to their large size, difficulty in removing blood, liver influx and low tissue penetration rate Has the disadvantage of indicating. To overcome this, methods have been developed that use antibody fragments such as F (ab), diabody, scFv, Fv and various single domain antibodies. Approaches using such antibody fragments have some limitations but are very useful. Efforts based on other protein scaffolds such as trinectins, anticalins and affibody ligands have been attempted to address stability issues such as hydrolysis and shelf-life.

On the other hand, a widely used vaccine delivery system is a method using viral liposomes. Viral liposomes may direct the delivery of nucleic acids to specific targets while at the same time functioning to protect against nucleic acid-degrading enzymes present in body fluids and cytoplasmic organelles. Tumor antigens are expressed in the background of semi-normal cells, as opposed to tumor cells. Nucleic acid vaccination offers the possibility of molecular immuno-physiological manipulation of antigen expression, which can be a useful means of cancer vaccine design. Examples of successfully delivering foreign DNA molecules into liposomes into cells include Nicolau and Sene, Biochim. Biophys. Acta , 721: 185-190 (1982) and Nicolau et al., Methods Enzymol. 149: 157-176 (1987). On the other hand, Lipofectamine (Gibco BRL) is the most used reagent for the transformation of animal cells using liposomes. In addition, a method of putting an anticancer agent in liposomes and delivering it to cancer cells has been used. However, the transport of anticancer drugs using liposomes has the disadvantage that the possibility of causing the leakage of anticancer drugs is very large.

The outer membrane of Gram-negative bacteria is very dynamic and produces vesicles during growth and releases them into the environment. Outer membrane vesicles (OMVs), also called blebs, contribute to the antigenic profile, including many outer membrane proteins and lipopolysaccharides. The antigenicity of these enveloped vesicles triggers immune responses in humans and animals, leading to diseases caused by Gram-negative bacteria (eg, meningococcal meningitis caused by Neisseria meningitidis ). Useful for treatment. However, numerous attempts at the envelope vesicle-based vaccines to effectively overcome the disadvantages of conventional encapsulated polysaccharide-based vaccines focus on treating a broader spectrum of bacterial infectious diseases.

As described above, since the conventional cancer treatment has toxicity and limitations, it is urgently required to develop more effective cancer treatment means.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have sought to develop a novel method for delivering anticancer agents specifically to cancer cells. As a result, the present inventors present an cancer-targeting ligand on the surface of outer membrane vesicles (OMVs) derived from Gram-negative bacteria to prepare an outer membrane vesicle that can specifically recognize cancer cells. By developing an anticancer drug delivery system using the same, the present invention has been completed by discovering that it can be applied to cancer treatment more efficiently and specifically.

It is an object of the present invention to provide outer membrane vesicles (OMVs) for cancer treatment.

Another object of the present invention to provide a pharmaceutical composition for anticancer.

Another object of the present invention is to provide a recombinant vector for cancer targeting.

It is another object of the present invention to provide a method for delivering an anticancer agent to a cell.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention is (a) outer membrane proteins (OMPs); (b) a cancer-targeting ligand bound to said envelope protein; And (c) provides outer membrane vesicles (OMVs) for the treatment of cancer comprising an anticancer agent.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the envelope membrane; And (b) provides a pharmaceutical composition for anticancer comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a composition comprising (a) (i) bacteria cytolysine A (Cly A); And (ii) an fusion protein composed of an affibody bound to the outer membrane protein; And (b) a promoter vector for cancer-targeting comprising a promoter sequence operatively linked to the fusion protein.

According to another aspect of the present invention, the present invention provides a method for delivering an anticancer agent to a cell, the method comprising contacting the cell with an outer membrane vesicle comprising the anticancer agent.

The present inventors have sought to develop a novel method for delivering anticancer agents specifically to cancer cells. As a result, the present inventors present the cancer-targeting ligand on the surface of the outer membrane vesicles derived from Gram-negative bacteria to prepare the outer membrane vesicles that can specifically recognize cancer cells, and develop an anticancer drug delivery system using the same. It has been found that it can be efficiently and specifically applied to cancer treatment.

Cancer is an invasion in which cells show unregulated growth through division above normal limits, expand and destroy surrounding tissues, and cause metastasis of cancer cells spreading through lymph or blood to other parts of the body. . The malignant characteristics of these cancers distinguish cancer from benign tumors that are self-limiting and do not penetrate or metastasize.

About 90-95% of cancers are caused by environmental factors and only 5-10% are caused by heredity. In general, the environmental factors that cause cancer include: tobacco (25-30%), eating and obesity (30-35%), infections (15-20%), radiation, stress, lack of exercise and environmental Contaminants. These environmental factors cause or increase abnormalities in the genetic material of the cell. The presence of cancer can be suspected based on symptoms or via radiation. However, a solid diagnosis of cancer requires microscopic analysis of biopsy specimens. Therefore, there is an urgent need for the design and development of new substances for target-specific anti-cancer therapies to improve the survival and quality of life of cancer patients.

The present invention provides a novel vaccine platform for the treatment of cancer, preferably outer membrane vesicles (OMVs) -based pharmaceutical compositions for anticancer.

Outer membrane vesicles (OMVs) are spherical bilayer vesicles typically having a diameter of 50-250 nm. Outer endoplasmic reticulum is characteristically produced by Gram-negative bacteria (Beveridge, 1999). Examples of bacterial species that produces outer membrane vesicles are Escherichia (Escherichia), Serratia marcescens (Serratia), nose, ceria (Neisseria), by a brush Russ (Haemophilus), Salmonella (Salmonella), Shigella (Shigella), Reggie ohnel La (Legionella), and a portion kolde Liao (Burkholderia), Helicobacter pylori (Helicobacter), Proteus (Proteus) or Pseudomonas (Pseudomonas). Outer endoplasmic reticulum is constantly separated from the surface of bacteria and grows in a wide variety of cells, but in general, lipopolysaccharides (LPS), periplasmic proteins, outer membrane proteins (OMPs), phospholipids, DNA , DNA-binding proteins and pathogenic factors (eg, alkaline phosphatase, phospholipase C (PLC), exotoxin A, DNase, hemolysine, proelastase, proteases and peptidoglycan hydrolases, etc. ).

Many of the components of the outer membrane vesicles described above can function as potent antigens. Outer membrane endoplasmic reticulum has been proposed to play an important role in a variety of pathogenic mechanisms, including cell transmembrane enzyme transport, DNA transport, bacterial adherence and immune system evasion (Horstman & Kuehn, 2000). In addition, several studies have identified that vesicles released by Neisseria gonorrhoeae and Haemophilus influenza can deliver DNA to receptor cells by releasing DNA from growing strains. Cherychia coli O157: H7 has been shown to release enveloped vesicles containing nucleic acids and cigar toxins (Kolling and Matthews, 1999). In addition, enveloped vesicles released from Gram-negative bacteria release many of the pathogenic factors necessary for the pathogenicity of bacteria into the environment around the target tissue to promote bacterial attachment and penetration, and have a direct effect on other bacteria.

In addition, bacterial OMVs are known to interact with eukaryotic cells and other bacteria and to mediate the transport of pathogenic factors through surface-expressed factors (Ketsy and Kuehn, 2004). For example, LPS-associated heat-sensitive enteroroxine (LT) on the surface of enterotoxigenic E. coli endoplasmic reticulum (ETEC) is a catalyst-active agent that causes internalization and infects eukaryotic cells through a caveolae. Carry the LT (Horstman &. Kuehn, 2000).

The present invention utilizes the above described enveloped vesicle-mediated delivery system for cancer treatment. The outer membrane vesicles of the present invention are (a) outer membrane proteins (OMPs); (b) a cancer-targeting ligand bound to said envelope protein; And (c) anticancer agents.

The term "envelope vesicles" herein means vesicles having a spherical lipid structure, including amphiphilic bilayers, preferably derived from Gram-negative bacteria. The envelope vesicles of the present invention may comprise various bioactive agents (eg, anticancer agents). In addition, the amphipathic bilayer comprises a lipid bilayer.

The envelope vesicles of the present invention have the following advantages: (a) small size (eg, 20-250 nm); (b) firm membrane structure (eg, unlike liposomes have a low likelihood of leakage); (c) effective loading of the anticancer agent (eg, loading of the anticancer agent into the endoplasmic reticulum spontaneously through co-mixing of the vesicle and anticancer agent); (d) ease of mass production and low production costs (eg, easy to separate / concentrate via bacterial culture); And (e) ease of storage (eg, convenient storage through lyophilization).

According to a preferred embodiment of the present invention, the size of the outer membrane vesicles of the present invention is 20-250 nm, more preferably 20-200 nm, even more preferably 20-150 nm, most preferably 20- 100 nm.

In addition, the outer membrane vesicles as an active ingredient of the present invention can be prepared as follows: (a) preparing a recombinant vector for cancer-targeting; (b) transforming said recombinant vector to bacteria; And (c) separating / concentrating the outer membrane vesicles from the culture medium of the bacteria.

According to a preferred embodiment of the present invention, the envelope membrane vesicles of the present invention can be prepared as follows: (a) envelope membrane protein of bacteria; And (ii) a fusion protein consisting of a cancer-targeting ligand bound to the outer membrane protein; And (b) preparing a recombinant vector comprising a promoter sequence operatively linked to the fusion protein; (b) transforming said recombinant vector to Gram-negative bacteria; And (c) separating / concentrating the outer membrane vesicles from the culture medium of the bacteria.

More preferably, (a) Cytolysine A (Cly A) of bacteria; And (ii) an fusion protein consisting of an affibody bound to said cytolysine A; And (b) preparing a recombinant vector comprising a promoter sequence operatively linked to the fusion protein; (b) transforming the recombinant vector to E. coli ; And (c) separating / concentrating the outer membrane vesicles from the E. coli culture.

Most preferably, (a) (iii) cytolysine A (Cly A) of bacteria; And (ii) a fusion protein consisting of affibody ( _HER2 ) capable of binding to human epidermal growth factor 2 ( _HER2 ) coupled with the cytolysin A; And (b) preparing a recombinant vector comprising a promoter sequence operatively linked to the fusion protein; (b) transforming the recombinant vector to E. coli ; And (c) separating / concentrating the outer membrane vesicles from the E. coli culture.

According to a preferred embodiment of the invention, the fusion protein of the invention further comprises a linker. The linker links bacterial outer membrane proteins (preferably cytolysine A) with cancer-targeting ligands (preferably, affibody _HER2 capable of binding to _HER2 ), as well as confirming expression of the fusion protein. It also functions as a marker for The linker of the present invention may use any linker used in the art, so long as it does not inhibit the function of the fusion protein (eg, cancer-targeting function), for example, c-Myc tag, 6 × His ( 6-Histidine (HA) tag, Hemagglutinin (HA) tag, S-tag, SBP tag, glutathione-S-transferase (GST) tag, maltose binding protein (MBP) tag and green fluorescence protein (GFP) tag It doesn't happen.

The amino acid sequence of the components constituting the fusion protein of the present invention is described in SEQ ID NO: 1 to SEQ ID NO: 5.

Preferably, the recombinant vector of the present invention comprises (i) a nucleotide sequence encoding the above-described expression target of the present invention (eg, fusion protein); (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on prokaryotic cells, preferably Gram-negative bacteria, to form RNA molecules, more preferably (iii) the invention described above Outer membrane protein of (a) (iii) bacteria of; And (ii) a nucleotide sequence encoding an amino acid sequence of a fusion protein consisting of a cancer-targeting ligand bound to the outer membrane protein, or a complementary nucleotide sequence thereof; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on a prokaryotic cell, preferably a Gram-negative bacterium, to form an RNA molecule, and even more preferably (iii) Cytolysine A of the bacterium of (a) (iii) of the invention; And (ii) a nucleotide sequence encoding an amino acid sequence of a fusion protein consisting of a cancer-targeting ligand bound to cytolysine A, or a complementary nucleotide sequence thereof; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on prokaryotic cells, preferably Gram-negative bacteria, to form RNA molecules, most preferably (i) Cytolysine A of the bacterium of (a) (iii) of the invention; And (ii) a nucleotide sequence encoding an amino acid sequence of a fusion protein consisting of affibody _HER2 capable of binding to HER2 bound to cytolysine A, or a nucleotide sequence thereof; (Ii) a recombinant expression vector operably linked to the nucleotide sequence of (iii) and comprising a promoter that acts on prokaryotic cells, preferably Gram-negative bacteria, to form RNA molecules.

Recombinant expression vectors of the invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as host cells, preferably prokaryotic cells. For example, prokaryotic cells include bacteria, more preferably Gram-negative bacteria.

According to a preferred embodiment of the present invention, prokaryotic cells that can be used as the host cell for transformation of the present invention are Gram-negative bacteria, more preferably, Escherichia, Serratia, Neisseria, Haemophilus, Salmonella, Shigella, Legionella, Bucolderia, Helicobacter, Proteus or Pseudomonas species, even more preferably Escherichia, Neisseria, Haemophilus, Salmonella or Shigella species, even more preferably Escherichia spp., Most preferably Escherichia coli .

As used herein, the term "promoter" refers to a DNA sequence that regulates the expression of a coding sequence or functional RNA. In the synthetic expression vector of the present invention, the expression-coding nucleotide sequence is operably linked to the promoter. As used herein, the term “operatively linked” refers to a functional binding between a nucleic acid expression control sequence (eg, a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence. Thus, the regulatory sequence will control the transcription and / or translation of the other nucleic acid sequence.

When the vector of the present invention is a prokaryotic cell as a host, powerful promoters capable of promoting transcription (e.g., tac promoter, lac promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 Promoters, amp promoters, recA promoters, SP6 promoters, trp promoters, T7 promoters, etc.), ribosomal binding sites for initiation of translation and transcription / detox termination sequences. Even more preferably, the host cell used in the present invention is an Escherichia species, most preferably E. coli . In addition, when E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)) can be used as regulatory sites. On the other hand, vectors that can be used in the present invention are plasmids (eg, pET28b, pRS316, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, etc.) often used in the art, phage (eg, λgt4λB, λ-Charon, etc.) , λΔz1, M13, etc.) or viruses (eg, SV40, etc.).

Preferably, the above-described expression target material includes a bacterial outer membrane protein and a cancer-targeting ligand as a fusion protein. According to a preferred embodiment of the present invention, the outer membrane protein of bacteria is ClyA, OmpA, OmpC, OmpF, OmpG, OmpN, OmpR, OmpW, OmpX, FecA, FhuA, FhuE, HlpA, HtrE, InvE, LamB, MltA, MltB, MltC, MltD, PgpB, PhoE, PhoP, RfaY, YbfM, YbiL or YeaF envelope membrane proteins, but are not limited thereto. Most preferably, the fusion protein of the present invention comprises an affibody _HER2 capable of binding to cytolysine A and HER2.

As used herein, the term “cancer-targeting ligand” is a cancer-targeting ligand presented on the surface of an outer endoplasmic reticulum derived from Gram-negative bacteria, and is a membrane protein overexpressed in cells of patients with cancer, tumors, or tumor-related diseases. For example, a ligand capable of binding to a receptor). Cancer-targeting ligands that may be used in the present invention include targeting moieties, aptamers, peptides, antibodies, antibody fragments, vitamins, drugs, nutraceuticals, carbohydrates, proteins, receptors, adhesion molecules, glycoproteins, sugar residues, articles Lycosaminoglycans or combinations thereof, but is not limited thereto.

According to a preferred embodiment of the present invention, the cancer-targeting ligand that can be used in the present invention is a targeting moiety, more preferably an affibody or a bipodal peptide binder, most preferably Is an affibody _HER2 or bipodal peptide binder capable of binding to _HER2 .

Epibody molecules are small proteins developed by Swedish biotechnology company Epibody AB and are based on 58 amino acid residues in the Z domain derived from the IgG-binding domain of staphylococcal protein A (SPA). (Nilsson, B, et al. , Protein Engineering , 1: 107-133 (1987)), selected from arbitrary molecular libraries using different interaction targets (W095 / 19374; W000 / 63243; Nord, K, et al. , Prot Eng, 8: 601-608 (1995); Nord, K, et al. , Nature Biotechnology, 15: 772-777 (1997)). The cysteine-free three-helix bundle domains of the Z domain are used as scaffolds, and epibody variants can be targeted to desired molecules using display technology (eg, phage display). Can be selected. Thus, epibodies can be prepared to bind to numerous target proteins or peptides with high affinity and mimic monoclonal antibodies and thus can be used for biochemical research and can be developed as powerful biopharmaceutical drugs. The simple and robust structure of Apibody with low molecular weight (7 kDa) is suitable for a wide range of applications. For example, the efficacy of epibodies can be applied to bioprocess- and laboratory-scale bioseparation processes (Nord, et al. , 2000, 2001; Gr, et al. , 2002), and receptor interactions. May be applied to inhibition (Sandstr, et al. , 2003). Thus, epibody ligands have the potential as therapeutic agents.

As used herein, the term “bipodal peptide binder (BPB)” refers to (i) parallel, antiparallel or parallel and antiparallel in which interstrand noncovalent bonds are formed. antiparallel) structure stabilizing region comprising amino acid strands; (Ii) a target binding region I and a target binding region II, each of which binds to both ends of the structural stabilization site and comprises randomly selected n and m amino acids, respectively; do.

A detailed description of the BPB is disclosed in WO 2010/047515 A2, which is incorporated herein by reference.

In BPB, the structure stabilization site includes parallel, antiparallel or parallel and antiparallel amino acid strands, interstrand hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi interactions. Protein structure motifs in which non-covalent bonds are formed by cation-pi interactions or combinations thereof. Non-covalent bonds formed by hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi interactions, cation-pi interactions, or combinations thereof between the strands are the rigidity of the structure stabilization site. Contribute to.

Optionally, there may be a covalent bond at the structured stabilization site. For example, disulfide bonds can be formed at the structured stabilization site to further increase the firmness of the structure stabilized site. The increase in firmness by such covalent bonds is given in consideration of the specificity and affinity of the bipodal peptide binder for the target.

According to a preferred embodiment of the invention, the amino acid strands of the structure stabilization site are linked by a linker. Linkers link parallel, antiparallel or parallel and antiparallel amino acid strands. For example, at least two strands (preferably two strands) aligned in parallel fashion, at least two strands (preferably two strands) aligned in antiparallel fashion, and at least three strands aligned in parallel and antiparallel fashion. The linker (preferably three strands) is connected by the linker. According to a preferred embodiment of the invention, the linker is a turn sequence or peptide linker. According to a preferred embodiment of the invention, the turn sequence is β-turn, γ-turn, α-turn, π-turn or ω-loop. Most preferably, the turn sequence used in the present invention is β-turn.

According to a preferred embodiment of the present invention, the structure stabilization site is a β-hairpin, a linker linked β-sheet or a linker leucine zipper, more preferably the structure stabilized site is a β-hairpin or linker linked β-sheet And most preferably β-hairpin. When a peptide having β-hairpin conformation is used as the structure stabilization site, most preferably a tryptophan zipper is used.

Random amino acid sequences are joined to both ends of the structure stabilization site described above. The random amino acid sequence forms target binding site I and target binding site II. One of the greatest features of the present invention is to prepare a peptide binder in a bipodal manner by connecting target binding site I and target binding site II to both ends of the structure stabilization site. Target binding site I and target binding site II cooperatively bind to the target, thereby greatly increasing the affinity for the target.

According to a preferred embodiment of the present invention, a functional molecule is additionally bound to the structure stabilization site, the target binding site I or the target binding site II (more preferably, the structure stabilization site, even more preferably the linker of the structure stabilization site) have. Examples of such functional molecules include, but are not limited to, labels, chemicals, biopharmaceuticals, cell transmembrane peptides (CPPs) or nanoparticles that generate detectable signals.

The HER2 proto-oncogene, also called neu, HER2 / neu or c-erbB-2, is a 185 kDa cell surface receptor protein (Hynes, NE, et al. , Biochim Biophys Acta , 1198: 165- 184 (1994). Normal cells express small amounts of HER2 protein in the cell membrane in a tissue-specific pattern. HER2 is known to form heterodimers with HER1 (EGFR), HER3 and HER4 in complex with ligands and thereby transmit activated HER2 receptor-transfer growth signals from the cell to the nucleus (Sundaresan. S., et al. , Curr Oncol Rep , 1: 16-22 (1999). In tumor cells, errors in the DNA replication system can result in the presence of multicopy genes on a single chromosome known as gene amplification. Amplification of the HER2 gene leads to increased transcription. As a result, increased HER2 mRNA levels result in an increase in HER2 protein levels resulting in overexpression of HER2 protein on the surface of tumor cells. Overexpression of HER2 is increased by 10 to 100 times the levels found in normal cells. Thus, amplification of the HER2 gene is involved in the transformation of normal cells into cancer phenotypes (Sundaresan. S., et al. , Curr Oncol Rep , 1: 16-22 (1999)).

According to the present invention, the outer membrane vesicles of the present invention comprise (a) cytolysine A, which is a bacterial outer membrane protein; (b) affibody _HER2 capable of binding to HER2 bound to cytolysine A; And (c) a linker (preferably, myc tack) which binds the cytolysine A and the epibody to the surface of the envelope membrane (see FIG. 1B). Therefore, it is possible to specifically bind to cancer cells or tumor cells overexpressing the HER2 protein through an affibody _HER2 capable of binding to HER2 presented on the surface of the outer membrane endoplasmic reticulum of the present invention.

The present invention can simply load the anticancer agent by mixing the outer membrane vesicles described above with a suitable anticancer agent. In the present invention, the anticancer agent to be loaded into the outer endoplasmic reticulum includes various anticancer agents used in the art, for example, to chemotherapeutic agents, anti-cancer small molecules, antibodies, or cancer cells. Specific biological agents.

According to a preferred embodiment of the present invention, chemotherapeutic agents which can be used in the present invention include various chemotherapeutic agents used in the art including alkylating agents, antimetabolites and intercalating agents. And, for example, docetaxel, 5-fluorouracil, 6-mercaptopurin, cytosine-arabinoside, fluda Fludarabine, methotrexate, doxorubicin, epirubicin, amekrin, cisplatin, carboplatin, procarbazine, mechloretta Mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin ( dactinomycin), the Norubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, transplatinum , Adriamycin, vincristin or vinblastin, most preferably docetaxel.

According to a preferred embodiment of the invention, the cancer which can be treated by the composition of the invention is breast cancer, oral cancer, prostate cancer, rectal cancer, non-small cell lung cancer, cleft lip cancer, liver cancer, lung cancer, anal cancer, kidney cancer, vulvar cancer , Oropharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, urethral cancer, small intestine cancer, biliary tract cancer, bladder cancer, ovarian cancer, laryngeal cancer, esophageal cancer, gallbladder cancer, colon cancer, head and neck cancer, Parathyroid cancer, penile cancer, vaginal cancer, thyroid cancer, pancreatic cancer, squamous cell carcinoma, Hogkin's lymphoma, leukemia-related diseases, mycosis fungoides or myelodysplastic syndrome.

In addition, the pharmaceutical composition of the present invention (a) a pharmaceutically effective amount of the outer membrane vesicles of the present invention described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the anticancer agent described above.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.0001-100 mg / kg per day on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to outer membrane vesicles (OMVs) comprising cancer-targeting ligands and uses thereof.

(b) the outer membrane vesicles of the present invention include (i) outer membrane proteins (OMPs); (Ii) cancer-targeting ligands bound to the outer membrane proteins; And (iii) an anticancer agent.

(c) The anticancer drug delivery system through the outer membrane endoplasmic reticulum of the present invention can more efficiently transport the anticancer agent into the cell compared to the conventional liposome-mediated method, and requires a low production cost because the mass production is very convenient. do.

(d) Thus, the composition using the outer membrane vesicles of the present invention can be applied to cancer treatment more efficiently and specifically compared to conventional methods for treating cancer (eg, liposome-mediated drug delivery systems).

1A is a diagram illustrating a vector map of a clyA-myc-Affibody _HER2 construct used in the present invention. In this experiment, we cloned cytolysine A (ClyA) into pET28b expression vector using T7 promoter, and then subcloned Affibody _HER2 containing myc tag to finally construct the cytolysine-myc-appibody (Affibody _HER2 ) construct. Completed.
Figure 1b is a schematic diagram showing the manufacturing process of the outer membrane vesicles for treatment of cancer of the present invention. The clyA-myc-Affibody _HER2 construct was transformed into E. coli to obtain an outer membrane vesicle containing the construct, and loaded with docetaxel, which is an anticancer agent, to obtain an outer membrane vesicle loaded with an anticancer agent.
Figure 2a is a photograph of the SDS-PAGE electrophoresis gel of clyA-myc-Affibody _HER2 protein overexpressed in E. coli through induction with IPTG.
Figure 2b is the result of Western blotting of the ClyA-myc-Affibody _HER2 protein expressed in the outer membrane vesicles purified from the E. coli . Western blotting results were performed using anti-myc monoclonal antibody (Abcam).
Figure 3a is the result of measuring the average size of the outer membrane vesicles expressed clyA-myc-Affibody _HER2 construct with ELS 8000, Figure 3b is the result of observing the morphology of the outer membrane vesicles by TEM.
Figure 4a is a test of HER2 binding whether the function of the affibody _HER2 using the isolated envelope vesicles properly. It was found that the HER2 protein adhered to the HER2 protein about 3.5 times better than the outer membrane vesicle (OMV) without the HER2 targeting (OMV-affibody _HER2 ).
5A-5C show the results of a cell binding test of the outer membrane vesicles of the present invention. Figure 5a is a result confirmed by immunostaining that the outer membrane vesicles of the present invention specifically bind the Affibody _HER2 mediated in SKOV3 cells overexpressing HER2 protein.
Figure 5b is a control experiment, the result of treating the outer membrane vesicles that do not express the clyA-myc-Affibody _HER2 construct in SKOV3 cells and the experimental method is as described above.
Figure 5c is the result of treating the outer membrane vesicles of the present invention to HELA cells not expressing the HER2 protein, the experimental method is the same.
Figure 5a-c shows that the outer membrane vesicles of the present invention specifically binds to the HER2 protein expressed on the cancer cell membrane.
Figure 6 measures the cell uptake rate of docetaxel of the outer membrane vesicles (OMV-affibody _HER2 ) with _HER2 targeting and outer membrane vesicles (OMV) without targeting. The outer membrane vesicle containing the targeting construct was better bound to BT-474, a cell overexpressing the HER2 protein, and it was confirmed that docetaxel enters the cells more than 2.5 times better.
Figure 7 measures the cytotoxicity of the outer membrane vesicles (OMV-affibody _HER2 ) with _HER2 targeting and outer membrane vesicles (OMV) without targeting. Targeted vesicles were found to kill BT-474 cells overexpressing HER2 protein about 10 times more effectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Fabrication of a ClyA Protein and Targeting Group Binding System

In order to obtain clyA gene from E. coli K12 w3110 strain, chromosomal DNA from w3110 strain was purified using cell sv mini kit (geneall). PCR reaction using two primers, forward primer (5'-CTAGGCTAGCATGACTGAAATCGTT-3 ') and reverse primer (5'-GATCGGATCCGACTTCAGGTACCTC-3'), PCR reaction (94 ° C, 5 minutes; 30 cycles-58 ° C, 30 seconds; 72 ° C) ClyA inserts were prepared through 1 minute 30 seconds and 94 ° C., 30 seconds, and purified using a PCR purification kit (GeneAll, Expin ^™ PCR SV). In order to link the clyA gene insert to the pET28b vector, restriction enzyme treatment was performed on the clyA gene insert and the pET28b vector. About 2 μg of insert DNA was reacted with Nhe I (NEB) and BamH I (NEB) for 4 hours and then purified using a PCR purification kit. In addition, about 2 μg of the pET28b vector was reacted with Nhe I (NEB) and BamH I (NEB) for 3 hours, and treated with CIP (Calf Intestinal Alkaline Phosphatase; NEB) for 1 hour and purified using a PCR purification kit. . Subsequently, the vector to insert was ligated at 18 ° C. for 10 hours using a T4 DNA ligase (Bioneer) for 10 hours, and then transformed into XL-1 competent cells and plated on kanamycin agar medium. Colonies grown on agar plate medium were inoculated in 5 ml of LB medium and incubated overnight at 37 ° C. at 200 rpm, followed by plasmid preparation using a plasmid preparation kit (GeneAll, Exprep ^TM Plasmid SV Mini). Was purified. To assess the success of cloning, sequencing was performed. Next, to put the affibody binding to HER2 after the clyA gene, the following primers were used: forward primer, 5'-AATGAATTCTCTTCCTCATCGGGTTCTTCCTCATCGGGTGAACAAAAACTCATC-3 '; And reverse primer, 5'-AATAAGCTTTCATTTTGGTGCTTGTGC-3 '. As a result, a HER2 epibody insert as a targeting gene was prepared. PET28b vector containing the insert and clyA was digested with restriction enzymes Eco RI (NEB) and Hind III (NEB), and then ligated using T4 DNA ligase. Cloning of the ClyA-myc-Affibody _HER2 construct was confirmed by sequencing.

Outer endoplasmic reticulum purification and ClyA-Affibody for cancer targeting _HER2 Expression experiment of

Western blotting was performed to determine whether ClyA-Affibody _HER2 is properly expressed in outer membrane vesicles (OMVs) recovered from E. coli incorporating ClyA-Affibody _HER2 prepared as described above. First, _ClyA-HER2 Affibody After the transfection the pET28b vector cloned a _ClyA-HER2 Affibody for purifying the expressed outer membrane vesicles in BL21 cells were plated on kanamycin agar. Colonies grown on agar plate medium were inoculated in LB medium (5 ml) containing kanamycin (25 μg / ml) and incubated overnight at 37 ° C. at 200 rpm. The culture was transferred to 50 ml LB medium (including 25 μg / ml kanamycin) and further incubated for 3 hours. The grown E. coli culture was added to 2 L LB medium (containing 25 μg / ml of kanamycin) and cultured to OD = 0.6-0.8. Then, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Gold biotechnology) was added and incubated for 12 hours with mixing at 37 rpm at 200 rpm. The precipitated cells were removed by centrifugation at 5,000 × g for 20 minutes and the supernatant was collected. Complete removal of E. coli using a 0.2 μm filter (NALGENE), collection of ClyA-Affibody _HER2 envelope vesicles, electrophoresis of proteins with SDS-PAGE, and Western blotting using anti-myc monoclonal antibody (Abcam) Was carried out.

OMV-affibody _HER2  Measurement of Size of Outer Vesicles

The size of the targeting envelope vesicles (OMV-affibody _HER2 ) was measured using ELS 8000 (Otsuka Electronics) and TEM (Philips Electronic Instrument Corp., Mahwah, NJ). Purified outer membrane vesicles were placed in a TEM grid, and then stained with 5% uranyl acetate (Sigma) to observe the shape and size of the outer membrane vesicles for targeting.

Targeting experiment for HER2 protein

In order to confirm whether the function of the affibody _HER2 is properly present, the HER2 binding test was performed using the isolated envelope vesicles. HER2 protein (5 [mu] g / ml; Bender Medsystems Inc) and control VEGF (5 [mu] g / ml; R & D system) were placed in a 96-well ELISA plate at 50 [mu] l / well, respectively, and left overnight at 4 [deg.] C. The following day, all wells were washed three times with 0.05% PBST and blotted for 2 hours at room temperature with 2% skim milk powder diluted with PBS, then all solutions were removed and washed three times with 0.05% PBST. 100 μl each of the targeting outer membrane vesicle (OMV-affibody _HER2 ) expressing ClyA-HER2_affibody and the control outer membrane vesicle were dispensed in the wells with HER2 protein and the well with VEGF protein, and 1 hour 30 minutes at 27 oC. I was stationed. After washing 5 times with 0.05% PBST solution, anti-myc monoclonal antibody (Abcam) was diluted 1: 1000 and reacted at 27 ° C for 1 hour. Then, after washing five times with 0.05% PBST, HRP-conjugated anti-IgG antibody (GE Healthcare) was diluted 1: 5,000 and reacted at 27 ° C for 1 hour. After washing 5 times with 0.05% PBST, 100 μl of TMB solution was dispensed to induce a color reaction, and then 100 μl of 1M HCl was added to stop the reaction. Absorbance was measured at 450 nm to determine whether the OMV-affibody _HER2 adhered better to the HER2 protein than the control OMV.

Targeting experiments on HER2 protein in cells

Human SKOV-3 cells and HeLa cells were incubated on the cover slip with 80% confluence, and then OMV-affibody _HER2 and control OMV were reacted with the cells at 4 ° C. for 1 hour. After washing three times with PBS, cells were fixed with 4% paraformaldehyde. OMV was then detected using anti-myc antibody (abcam) and anti-secondary antibody-FITC, and cells were observed using Leica TCS SP2 AOBS confocal microscopy (Mannheim, Germany).

Injecting anticancer drugs into the outer membrane vesicles

In order to confirm the anticancer effect using the outer membrane endoplasmic reticulum, docetaxel (Dotataxel; Toronto Research Chemicals Inc), which is an anticancer agent, was filled in the outer membrane vesicle. First, in order to obtain OMV-affibody _HER2 , E. coli was incubated in 50 ml LB medium, and then added to 2 L LB containing kanamycin (25 µg / ml) and cultured to OD = 0.6-0.8. 0.2 mM IPTG was added to the culture solution and incubated at 37 ° C. at 200 rpm for 12 hours. After removing the precipitated cells by centrifugation at 5,000 x g for 20 minutes, all the supernatant was collected. E. coli was completely removed using a 0.2 μm filter, the outer membrane vesicles were collected and concentrated, and centrifuged at 40,000 rpm for one hour to collect the outer membrane vesicles for targeting. 100 μg of the outer membrane vesicles and 100 μg of docetaxel dissolved in 20% ethanol were mixed, and then predocetaxel was removed using a 0.2 μm filter and a Superdex 75 column (GE Healthcare). In order to measure the amount of docetaxel loading, 2% Triton X-100 (USB Corp.) was dissolved in the outer membrane vesicles, and docetaxel was separated with 90% ethyl acetate, and then the amount of docetaxel was measured using HPLC.

Cell absorption experiment

In order to measure the cellular uptake of HER2_affibody-expressed outer membrane vesicles (OMV-affibody _HER2 ) and control original outer membrane vesicles, the BTF474 breast cancer cell line, HER2-positive cell line, and the MCF-7 breast cancer cell line, HER2-negative cell line (ATCC). Cell uptake experiments were conducted in (Manassas, VA). BT-474 cells and MCF-7 cells were incubated for 24 hours in 5 × 10 ⁴ cells / well in 96-well plates, followed by adding HER2_affibody envelope vesicles containing 100 ng / ml of docetaxel and the original envelope vesicles at 37 ° C. The reaction was further reacted for 2 hours at and washed 4 times with PBS. To measure the amount of docetaxel in the cells, trypsin (Invitrogen ^TM ) was added to collect the cells, 0.1% SDS was added to lyse the cells, and ACN (Fisher Scientific) was added. Then, the amount of docetaxel was measured using HPLC in the supernatant obtained by centrifugation at 13,000 rpm for 5 minutes.

Cytotoxicity Experiment

For cytotoxicity experiments of HER2 targeting envelope vesicles loaded with docetaxel, MTT tests were performed using BT-474 breast cancer cells, HER2-positive cell lines, and MCF-7 breast cancer cells, HER2-negative cell lines. BT-474 cells and MCF-7 cells were incubated for 24 hours in 5 × 10 ³ cells / well in 96-well plates, followed by 1000 ng / ml, 100 ng / ml, 10 ng / ml or 1 ng / ml The outer membrane vesicles (OMV-affibody _HER2 ) containing HER2_affibody and the original outer membrane vesicles as a control group were added, and after 48 hours, MTT solution (Sigma) was treated to measure the degree of cytotoxic inhibition.

Experiment result

Creating a Combined System of ClyA and Targeting Groups

ClyA protein is a protein that is abundantly present in the OMV and forms a multimer and is distributed on the outer membrane. The target group can be linked to the c-terminus to express foreign proteins or peptides on the surface of the outer membrane vesicle. In particular, the outer membrane endoplasmic reticulum that can target cancer by combining the affibody protein that binds specifically to HER2 was produced as shown below. Myc-tack is a fused sequence to test expression experiments.

OMV Purification and Expression Testing in OMV

In order to test the expression level of ClyA-myc-Affibody _HER2 in OMV, the purified OMV was subjected to western blot. The first antibody was tested using anti-myc monoclonal antibody and the second antibody was tested using anti-IgG-HRP polyclonal antibody. As can be seen in Figure 1, it can be seen that there are a lot of ClyA-myc-Affibody _HER2 induced by IPTG in the OMV. Therefore, this experiment shows that ClyA-myc-Affibody _HER2 exists in OMV.

OMV-affibody _HER2 Size measurement

ELS 8000 was used to measure the size of the outer membrane vesicles of OMV-affibody _HER2 . As a result, the size of the outer membrane vesicles of the present invention was 43 ± 10 nm. In order to know the exact size and shape of the outer vesicles, TEM was performed. As a result of the TEM photograph, it was confirmed that the outer membrane vesicles of the present invention had a round shape at 20-50 nm.

Targeting experiment

HER2 binding tests were performed to determine whether affibody _HER2 administered using the isolated envelope vesicles was present and functioning properly. As can be seen in the figure, compared to the outer membrane vesicles (OMV-affibody _HER2 ) without HER2 targeting (OMV) alone was confirmed that the binding to the HER2 protein about 3.5 times better. In addition, it can be seen that the outer membrane vesicle (OMV-affibody _HER2 ) with HER2 targeting does not adhere to the VEGF protein as a control.

Intracellular binding test

The HER2-positive cell line SKOV-3 and the HER2-negative cell line HeLa cells were used to determine whether OMV-affibody _HER2 specifically binds to HER2. As can be seen in the figure, it was found that OMV-Affibody _HER2 was more attached to SKOV-3 than OMV. In addition, HeLa cells, which are HER2-negative cell lines, did not adhere to OMV-affiobdy _HER2 . Through this, it was confirmed that HER2 can be specifically recognized on cells.

Cellular uptake experiment

The cell uptake rate of docetaxel of the outer membrane vesicles (OMV-affibody _HER2 ) with HER2 targeting and the outer membrane vesicles (OMV) without targeting was measured. As shown in the figure, targeting outer membrane endoplasmic reticulum was confirmed that more than 2.5 times the docetaxel is better into the cells in BT-474 HER2-positive cells.

Cytotoxicity experiment

Cytotoxicity of _HER2 -targeted envelope vesicles (OMV-affibody _HER2 ) and non-targeted envelope vesicles (OMV) was measured. As can be seen in the figure, targeting membrane vesicles can be confirmed to kill cancer cells about 10 times more effectively in BT-474, a HER2-positive cell line.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> GIST <120> Pharmaceutical Compositions for Treatment of Cancer Comprising          Outer Membrane Vesicles as an Active Ingredient <130> PN100502 <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 303 <212> PRT <213> Escherichia coli <400> 1 Met Thr Glu Ile Val Ala Asp Lys Thr Val Glu Val Val Lys Asn Ala   1 5 10 15 Ile Glu Thr Ala Asp Gly Ala Leu Asp Leu Tyr Asn Lys Tyr Leu Asp              20 25 30 Gln Val Ile Pro Trp Gln Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser          35 40 45 Arg Phe Lys Gln Glu Tyr Ser Gln Ala Ala Ser Val Leu Val Gly Asp      50 55 60 Ile Lys Thr Leu Leu Met Asp Ser Gln Asp Lys Tyr Phe Glu Ala Thr  65 70 75 80 Gln Thr Val Tyr Glu Trp Cys Gly Val Ala Thr Gln Leu Leu Ala Ala                  85 90 95 Tyr Ile Leu Leu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Gln             100 105 110 Lys Asp Ile Leu Ile Lys Val Leu Asp Asp Gly Ile Thr Lys Leu Asn         115 120 125 Glu Ala Gln Lys Ser Leu Leu Val Ser Ser Gln Ser Phe Asn Asn Ala     130 135 140 Ser Gly Lys Leu Leu Ala Leu Asp Ser Gln Leu Thr Asn Asp Phe Ser 145 150 155 160 Glu Lys Ser Ser Tyr Phe Gln Ser Gln Val Asp Lys Ile Arg Lys Glu                 165 170 175 Ala Tyr Ala Gly Ala Ala Ala Gly Val Val Ala Gly Pro Phe Gly Leu             180 185 190 Ile Ile Ser Tyr Ser Ile Ala Ala Gly Val Val Glu Gly Lys Leu Ile         195 200 205 Pro Glu Leu Lys Asn Lys Leu Lys Ser Val Gln Asn Phe Phe Thr Thr     210 215 220 Leu Ser Asn Thr Val Lys Gln Ala Asn Lys Asp Ile Asp Ala Ala Lys 225 230 235 240 Leu Lys Leu Thr Thr Glu Ile Ala Ala Ile Gly Glu Ile Lys Thr Glu                 245 250 255 Thr Glu Thr Thr Arg Phe Tyr Val Asp Tyr Asp Asp Leu Met Leu Ser             260 265 270 Leu Leu Lys Glu Ala Ala Lys Lys Met Ile Asn Thr Cys Asn Glu Tyr         275 280 285 Gln Lys Arg His Gly Lys Lys Thr Leu Phe Glu Val Pro Glu Val     290 295 300 <210> 2 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 2 Val Asp Asn Lys Phe Asn Lys Glu Met Arg Asn Ala Tyr Trp Glu Ile   1 5 10 15 Ala Leu Leu Pro Asn Leu Asn Asn Gln Gln Lys Arg Ala Phe Ile Arg              20 25 30 Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala          35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys      50 55 <210> 3 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> myc_affibody <400> 3 Glu Gln Lys Leu Ile Ser Glu Glu Asp Val Asp Asn Lys Phe Asn Lys   1 5 10 15 Glu Met Arg Asn Ala Tyr Trp Glu Ile Ala Leu Leu Pro Asn Leu Asn              20 25 30 Asn Gln Gln Lys Arg Ala Phe Ile Arg Ser Leu Tyr Asp Asp Pro Ser          35 40 45 Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln      50 55 60 Ala pro lys  65 <210> 4 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> Linker_myc_affibody <400> 4 Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Glu Gln Lys Leu Ile Ser   1 5 10 15 Glu Glu Asp Val Asp Asn Lys Phe Asn Lys Glu Met Arg Asn Ala Tyr              20 25 30 Trp Glu Ile Ala Leu Leu Pro Asn Leu Asn Asn Gln Gln Lys Arg Ala          35 40 45 Phe Ile Arg Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu      50 55 60 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys  65 70 75 <210> 5 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> ClyA_linker_myc_affibody <400> 5 Met Thr Glu Ile Val Ala Asp Lys Thr Val Glu Val Val Lys Asn Ala   1 5 10 15 Ile Glu Thr Ala Asp Gly Ala Leu Asp Leu Tyr Asn Lys Tyr Leu Asp              20 25 30 Gln Val Ile Pro Trp Gln Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser          35 40 45 Arg Phe Lys Gln Glu Tyr Ser Gln Ala Ala Ser Val Leu Val Gly Asp      50 55 60 Ile Lys Thr Leu Leu Met Asp Ser Gln Asp Lys Tyr Phe Glu Ala Thr  65 70 75 80 Gln Thr Val Tyr Glu Trp Cys Gly Val Ala Thr Gln Leu Leu Ala Ala                  85 90 95 Tyr Ile Leu Leu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Gln             100 105 110 Lys Asp Ile Leu Ile Lys Val Leu Asp Asp Gly Ile Thr Lys Leu Asn         115 120 125 Glu Ala Gln Lys Ser Leu Leu Val Ser Ser Gln Ser Phe Asn Asn Ala     130 135 140 Ser Gly Lys Leu Leu Ala Leu Asp Ser Gln Leu Thr Asn Asp Phe Ser 145 150 155 160 Glu Lys Ser Ser Tyr Phe Gln Ser Gln Val Asp Lys Ile Arg Lys Glu                 165 170 175 Ala Tyr Ala Gly Ala Ala Ala Gly Val Val Ala Gly Pro Phe Gly Leu             180 185 190 Ile Ile Ser Tyr Ser Ile Ala Ala Gly Val Val Glu Gly Lys Leu Ile         195 200 205 Pro Glu Leu Lys Asn Lys Leu Lys Ser Val Gln Asn Phe Phe Thr Thr     210 215 220 Leu Ser Asn Thr Val Lys Gln Ala Asn Lys Asp Ile Asp Ala Ala Lys 225 230 235 240 Leu Lys Leu Thr Thr Glu Ile Ala Ala Ile Gly Glu Ile Lys Thr Glu                 245 250 255 Thr Glu Thr Thr Arg Phe Tyr Val Asp Tyr Asp Asp Leu Met Leu Ser             260 265 270 Leu Leu Lys Glu Ala Ala Lys Lys Met Ile Asn Thr Cys Asn Glu Tyr         275 280 285 Gln Lys Arg His Gly Lys Lys Thr Leu Phe Glu Val Pro Glu Val Gly     290 295 300 Ser Glu Phe Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Glu Gln Lys 305 310 315 320 Leu Ile Ser Glu Glu Asp Val Asp Asn Lys Phe Asn Lys Glu Met Arg                 325 330 335 Asn Ala Tyr Trp Glu Ile Ala Leu Leu Pro Asn Leu Asn Asn Gln Gln             340 345 350 Lys Arg Ala Phe Ile Arg Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala         355 360 365 Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys     370 375 380

Claims (16)

(a) outer membrane proteins (OMPs); (b) a cancer-targeting ligand bound to said envelope protein; And (c) outer membrane vesicles (OMVs) for treating cancer, including an anticancer agent.
2. The envelope vesicles of claim 1, wherein the envelope vesicles are derived from Gram-negative bacteria.
The outer membrane endoplasmic reticulum according to claim 2, wherein the Gram-negative bacteria is Escherichia coli .
The envelope membrane of claim 1, wherein the envelope protein is cytolysin A.
The envelope membrane of claim 1, wherein the cancer-targeting ligand is an affibody or a bipodal peptide binder.
6. The envelope membrane of claim 5, wherein the epibody is an affibody _HER2 capable of binding to human epidermal growth factor receptor 2 ( _HER2 ).
6. The outer membrane vesicles of claim 5, wherein the bonding is via a linker.
The outer membrane vesicles of claim 1, wherein the anticancer agent is docetaxel.
The envelope membrane of claim 1, wherein the envelope membrane has a size of 20-100 nm.
The method of claim 1, wherein the cancer is breast cancer, oral cancer, prostate cancer, rectal cancer, non-small cell lung cancer, cleft lip cancer, liver cancer, lung cancer, anal cancer, kidney cancer, vulvar cancer, oropharyngeal cancer, nasal / sinus cancer (nasal cavity and paranasal sinus cancer), nasopharyngeal cancer, urethral cancer, small intestine cancer, biliary tract cancer, bladder cancer, ovarian cancer, laryngeal cancer, esophageal cancer, gallbladder cancer, colon cancer, head and neck cancer, parathyroid cancer, penile cancer, vaginal cancer, thyroid cancer, pancreatic cancer, Outer endoplasmic reticulum characterized by being squamous cell carcinoma, Hodgkin's lymphoma, leukemia-related disease, mycosis fungoides or myelodysplastic syndrome.
(a) a pharmaceutically effective amount of the outer membrane vesicles of any one of claims 1 to 10; And (b) a pharmaceutically acceptable carrier.
(a) (iii) Cytolysine A (Cly A) of bacteria; And (ii) an fusion protein composed of an affibody bound to the outer membrane protein; And (b) a promoter vector operatively linked to the fusion protein.
The recombinant vector of claim 12, wherein the fusion protein further comprises a linker.
The recombinant vector of claim 12, wherein the epibody is an affibody _HER2 capable of binding to human epidermal growth factor receptor 2 ( _HER2 ).
A method for delivering an anticancer agent to a cell comprising contacting the outer membrane endoplasmic reticulum of any one of claims 1 to 10 comprising an anticancer agent.
The method of claim 15, wherein the anticancer agent is docetaxel.

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