KR20150010882A - Fusion protein comprising annexin A2 binding protein, p53 and p18, and composition comprising the fusion protein for preventing or treating cancer - Google Patents

Abstract

The present invention relates to a fusion protein having an annexin A2 binding protein, a p53 protein, and a p18 protein bound, and to a pharmaceutical composition including the fusion protein for preventing or treating a cancer. Provided in an embodiment of the present invention is a fusion protein including an annexin A2 binding protein, a p53 protein or a fragment of the same, and a p18 protein. The fusion protein can additionally include one or more selected from the group consisting of an in vitro stabilization protein, a membrane transfer sequence (MTS) domain, a nucleus-cytoplasm signal domain, ATT, and STOPx2.

Description

[0001] The present invention relates to a fusion protein comprising annexin A2 binding protein, p53 protein and p18 protein, and a composition for preventing or treating cancer comprising the fusion protein, annexin A2 binding protein, p53 protein and p18 protein, cancer}

A fusion protein in which an annexin A2 binding protein, a p53 protein and a p18 protein are bound, and a pharmaceutical composition for preventing or treating cancer comprising the fusion protein.

In order for the cells to proliferate, they cycle through a series of steps through the G1 phase, the DNA replicator (S phase), the G2 phase and the cell divider (M phase), which is called the cell cycle . A cell divider is a practical stage in which cells multiply through mitosis and cytoskeleton, DNA is replicated in the DNA replicator, and the number of cells does not change. Therefore, in the cell cycle, the DNA replicators except the cell divider, G1 and G2 are called interphase. The G1 and G2 groups contain checkpoints that control the cell cycle, which plays an important role in preventing cells from abnormally proliferating. After confirming that the conditions for progressing from the checkpoint to the next stage of the cell cycle are met, the DNA replicator or cell divider progresses if all conditions are met. Failure to properly control cell proliferation at these checkpoints leads to abnormal cell proliferation, such as cancer.

Activation of the cyclin-dependent kinase (CDK) protein is required to cross the G1 checkpoint and to mitigate past the DNA replicator, G2 checkpoint. CDKs can not have activity alone and must be associated with cyclin proteins to have kinase activity. The cyclin-CDK complex induces cell division by phosphorylating proteins involved in cell proliferation. The proteins that induce DNA replication are mainly Cyclin D / E, CDK 4/6, and CDK 4/6 phosphorylates the Rb (retinoblastoma) protein, resulting in the transcriptional activity of the transcription factor E2F. The proteins that induce cell division are mainly Cyclin A / B, CDK 2/1, and their Cyclin-CDK complexes are referred to as MPF (M-phase promoting factor). Once the mitosis is complete or DNA replication is complete, no further kinase activity is required, and the cyclin in the cyclin-CDK complex is degraded and CDK activity is lost.

There are many factors that regulate the activity of the cyclin-CDK complex for cell cycle control. Typically, there is a p21 protein that acts when DNA is damaged at the G1 checkpoint. The p21 gene is expressed by p53, a tumor suppressor gene that is activated when DNA is damaged. p21 binds to a cyclin-CDK complex that induces DNA replication, thereby inhibiting the kinase activity of CDK4 / 6/2, thereby preventing phosphorylation of the Rb protein. As a result, the cells stay in the G1 phase and have time to repair the damaged DNA. If the DNA damage is so severe that the cell is unacceptable, p53 expresses the Bax protein gene, an apoptosis-inducing gene. In other words, in the regulation of cell proliferation, the action of p53 is either growth arrest by expressing the p21 gene, or p53 protein coupled with p53-binding protein ASPP protein expresses Bax gene to induce apoptosis And induction of apoptosis (induction of apoptosis can be divided into two. Apoptosis is also called programmed cell death, and when cell death is more helpful at the individual level, such as when the cell is no longer needed in the tissue, when it has been infected with a virus, or when it has been transformed into cancer cells, It is a mechanism.

CDK inhibitors that regulate the cell cycle are classified into two classes: CIP (CDK inhibitor protein) family and INK4 (Inhibitors of cyclin-dependent kinase 4). CIP has p21 and p27. INK4 mainly inhibits the activity of CDK 4, 6 and has p18, p16, p8, p19.

In order to control cell proliferation, the balance between the tumor suppressor gene and the proto-oncogene must be maintained. Mutation of the p53 protein gene, which is a tumor suppressor gene, can not induce apoptosis of abnormal cells, resulting in abnormal cell proliferation. In addition, the precancerous gene, which is a normal cell proliferative factor, induces abnormal cell proliferation such as cancer cells even if it becomes a cancer gene by mutation (gene substitution by virus, etc.) or overexpression. Cancer genes produce abnormal growth factors, intracellular signaling proteins and cause false signal transduction, so cell proliferation occurs without external proliferation signal.

In view of this fact, in order to inhibit cancer caused by abnormal cell proliferation, development of an anticancer drug using various factors controlling cell cycle is required.

One embodiment provides a fusion protein comprising anexin A2 binding protein, p53 protein or fragment thereof, and p18 protein.

Another embodiment provides a polynucleotide encoding said fusion protein.

Another embodiment provides a recombinant vector comprising said polynucleotide.

Another embodiment provides a cell transformed with said recombinant vector.

Another embodiment provides a method of producing a fusion protein comprising culturing the transformed cells.

Another embodiment provides a pharmaceutical composition for preventing and / or treating cancer comprising the fusion protein as an active ingredient.

Another embodiment provides a method of treating cancer comprising administering to the individual a therapeutically effective amount of the fusion protein.

An example of the present invention provides a fusion protein comprising anexin A2 binding protein, p53 protein or fragment thereof, and p18 protein. The fusion protein may comprise at least one selected from the group consisting of an in vitro stabilization protein, a membrane transfer sequence (MTS) domain, a nucleus-cytoplasm signal domain, ATT and STOPx2 May be further included.

Another example provides a polynucleotide encoding the fusion protein.

Another example provides a recombinant vector comprising the polynucleotide.

Another example provides a cell transformed with the recombinant vector.

Another example provides a method for producing a fusion protein comprising culturing the transformed cells.

Yet another example provides a pharmaceutical composition for preventing and / or treating cancer comprising the fusion protein as an active ingredient. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent and / or excipient in an amount normally used.

As described above, CDK (cyclin-dependent kinase) protein plays a major role in cancer development, and its inhibitors include CIP (CDK inhibitor protein) system protein and INK4 (Inhibitors of cyclin-dependent kinase 4) system protein. The fusion protein according to one embodiment of the present invention is a fusion protein of a p53 protein that regulates the expression of CIP lineage protein and a p18 protein that belongs to INK4 lineage protein and exhibits both CIP system protein activity and INK4 protein activity, / / Therapeutic effect can be obtained.

According to one embodiment, the p53 protein may be a polypeptide having an amino acid sequence provided by the NCBI accession number NP_000537 or a fragment thereof. As used herein, the p53 protein is used to mean both a full-length protein and a fragment thereof, unless otherwise noted.

Also, the p53 protein may be a transcriptionally active domain of the p53 protein having the amino acid sequence of SEQ ID NO: 1 (Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn).

The p53 protein fragment having the amino acid sequence of SEQ ID NO: 1 means a transcription activity domain comprising the 18th to 26th amino acid sequences of the human p53 protein, which is a site binding to Mdm2 in the p53 protein. The transcriptional activation domain of p53 binds to Mdm2 which has a function of p53 degradation, thereby greatly increasing the stability of p53 in vivo. By the action of p53, p21 gene encoding the CDK inhibitor protein is expressed to temporarily stop cell proliferation p53, which binds to the p53-binding protein ASPP (apoptosis-stimulating protein of p53), plays a role in inducing cell death by expressing the Bax gene. In addition, the tumor suppressor p18 protein plays a role in inhibiting the activity of CDK4,6. Therefore, a fusion protein in which a transcription activation domain of p53 and a p18 protein are combined and a pharmaceutical composition containing the fusion protein as an active ingredient can be very useful as a novel anticancer agent.

According to one embodiment, the p18 protein may be a polypeptide having the amino acid sequence of SEQ ID NO: 2. In addition, for the purpose of improving the solubility, the p18 protein can be used by modifying (substituting, deleting, addition, deletion, etc.) some amino acid residues unless it affects the whole protein activity.

The sequence of the p53 protein and the p18 protein in the fusion protein is not limited, and includes both the N-terminal p53 protein-p18 protein-C terminal end or the N terminal p18 protein-p53 protein-C terminal form. In one embodiment, the fusion protein in which the p53 protein fragment of SEQ ID NO: 1 and the p18 protein of SEQ ID NO: 2 are combined may have the amino acid sequence of SEQ ID NO: 3.

In addition, the fusion protein of the present invention includes an Annexin A2 binding protein (Annexin A2BP). The annexin A2 binding protein binds to annexin A2, which is specifically expressed in cancer cells, so that cancer cells can be targeted so that the fusion protein selectively acts on cancer cells. The annexin A2 protein may be exemplified by a protein having the amino acid sequence of SEQ ID NO: 4, but the scope of the present invention is not limited thereto. As long as the cancer cell targeting activity can be achieved, the annexin A2 binding protein fragment, Or a mutant may be used.

The annexin A2 binding protein in the fusion protein may be located at the N-terminus or C-terminus of the p53 and p18 fusions. For example, the fusion protein of the present invention includes a fusion protein comprising annexin A2 binding protein, p53 protein and p18 protein in the order of N-terminal to C-terminal; A fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of N-terminal to C-terminal; a fusion protein comprising the p53 protein, the p18 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal; And a fusion protein comprising the p18 protein, the p53 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal.

In a preferred embodiment, the fusion protein comprises an in vitro stabilization protein, a membrane transfer sequence (MTS) domain, a nucleus-cytoplasm signal domain, and an in vivo stabilization protein (in vivo stabilization protein) may be further included.

In this case, the position of the added polypeptide in the fusion protein is not limited, and each of the added one or more polypeptides can be independently included in the N-terminus, C-terminus, or between the p53 protein and the p18 protein of the fusion protein.

Preferably, the fusion protein comprises an annexin A2 binding protein, a p53 protein and a p18 protein in the order of N-terminal to C-terminal, wherein the fusion protein comprises an in vitro stabilization protein in the N-terminal direction of the p53 protein, A transmembrane domain, and a nucleus-cytoplasmic signal domain, or comprises at least one selected from the group consisting of transmembrane domain, nuclear-cytoplasmic signal domain and in vivo stabilizing protein in the C-terminal direction of p18 protein , And < / RTI >

Also preferably, the fusion protein is a fusion protein comprising an annexin A2 binding protein, a p18 protein and a p53 protein in the order of N-terminal to C-terminal, wherein the fusion protein comprises an in vitro stabilization protein, A transmembrane domain, a transmembrane domain, a transmembrane domain, a transmembrane domain, a transmembrane domain, and a nuclear-cytoplasmic signal domain, or comprises at least one selected from the group consisting of transmembrane domain, Or a combination thereof.

As used herein, the term "in vitro stabilization protein" refers to a protein for enhancing the solubility and stability of the fusion protein in vitro, i.e., when experimentally purifying the fusion protein . The ex-vivo stabilizing protein, as part of the fusion protein, should not induce immunogenicity in vivo.

In one embodiment, when the in vitro stabilizing protein is introduced into the fusion protein, it is preferably, but not limited to, located in the N-terminal direction of the fusion protein. Also preferably, the in vivo stabilizing protein may be additionally located between p18 and p53 in addition to both ends of the fusion protein.

According to one embodiment, the ex vivo stabilizing protein may be, but is not limited to, ubiquitin or ubiquitin-like protein.

Ubiquitin (Ub) is the most conserved protein found in nature, consisting of 76 amino acid sequences, and is a water-soluble protein that exhibits perfect homology between evolutionary species such as insects, trout, and humans. In addition, ubiquitin is known to be stable to changes in pH, not easily denatured at high temperatures, and stable to proteases. Thus, ubiquitin can improve the insolubility of the fusion protein.

The ubiquitin-like protein (Ubl) may be selected from the group consisting of wild-type ubiquitin, wild-type ubiquitin-like protein, mutant ubiquitin and mutant ubiquitin-like protein. The mutant ubiquitin means that the amino acid sequence of the wild-type ubiquitin is replaced with another amino acid sequence. For example, ubiquitin substituted with Lys of wild type ubiquitin (SEQ ID NO: 5) and / or wild type ubiquitin C- (I.e., the 76th Gly of the ubiquitin wild-type polypeptide is replaced by Ala). According to one embodiment, in the mutant ubiquitin in which the Lys of the wild-type ubiquitin is substituted with Arg, the substitution is preferably one or more selected from Lys existing at 6th, 11th, 27th, 29th, 33th, 48th and 63rd positions of the wild type ubiquitin , And the substitution can be performed independently or in combination with the position of the Lys.

According to one embodiment, the ubiquitin-like protein is a protein whose characteristics are similar to those of ubiquitin, for example, a protein selected from the group consisting of Nedd8, SUMO-1, SUMO-2, NUB1, PIC1, UBL3, UBL5 and ISG15 Or more, but is not limited thereto.

According to one embodiment, the ubiquitin or ubiquitin-like protein may comprise an amino acid sequence which is cleavable by a protease at the C-terminus or an amino acid sequence which is not cleaved by a protease. Amino acid sequences cleavable by the protease can be identified through a search database known in the art. For example, the protease that is searched at http://www.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html and its cleavable amino acid sequence can be used. When the cleavable amino acid sequence is included, the fusion protein is permeabilized into a cell, and then the ubiquitin or ubiquitin-like protein is cleaved by the intracellular protease, and the protein containing the annexin A2 binding protein, the p53 protein and the p16 protein Allowing the fusion protein to function in the cell. After the cleavage, the fusion protein may include a transmembrane sequence domain and / or a nucleus-cytoplasmic signal domain, but they do not affect the function of the fusion protein because the length of the polypeptide is very short. Even if the ubiquitin or ubiquitin-like protein is not cleaved by the intracellular protease, the ubiquitin or ubiquitin-like protein is not safe in the living body since it does not have immunogenicity, and since it does not contain cysteine and does not fold, And does not affect the ability of the fusion protein to exert its function in the cell.

The term "transmembrane (membrane transfer)" is in vitro (in vitro) and / or in vivo (in vivo < / RTI > of the fusion protein to be delivered into the cell or into the nucleus.

In addition, "membrane transfer sequence (MTS) domain" refers to a polypeptide having an amino acid sequence that can pass through the cell membrane of the phospholipid bilayer as such. The membrane permeable sequence domain has a single hydrophobic region at its N-terminus, forms a helix structure, exhibits flexibility, and has a relatively short length of amino acids (7 to 17 amino acids) . Thus, the physical properties of the transmembrane sequence domain usually exhibit hydrophobicity. According to one embodiment, the membrane-permeable sequence domain can be any polypeptide as long as it has an amino acid sequence that can pass through the cell membrane of the phospholipid bilayer, and is not particularly limited. However, the polypeptide transmembrane domain having the amino acid sequence of SEQ ID NO: 6 Lt; / RTI >

In one embodiment, when the transmembrane sequence domain is introduced into the fusion protein, it is preferably, but not exclusively, located in the N-terminal direction of the fusion protein.

The term "nucleus-cytoplasm signal domain" is interpreted to mean a polypeptide sequence that serves to transport a fusion protein into the interior of the nucleus or to transport it outside of the nucleus. According to one embodiment, the nuclear-cytoplasmic signal domain may be a nucleus location sequence (NLS) domain or a nucleus export sequence (NES) domain. That is, in order to transfer the fusion protein into the nucleus, the fusion protein may include NLS in order to include NLS in the fusion protein and to allow the fusion protein to remain in the cytoplasm.

The NLS domain is a protein that is transported from the cytoplasm to the nucleus and the NES domain is a protein that is transported from the nucleus to the cytoplasm. The above domain means a polypeptide having an amino acid sequence that can pass through the nuclear membrane, Is not particularly limited. The polypeptide that can be used as the NLS domain can be, for example, but not limited to, KKKRK (SEQ ID NO: 7), PKKKRKV (SEQ ID NO: 8), KRPAATKKAGQAKKKK (SEQ ID NO: 9), and the like.

The nuclear-cytoplasmic signal domain in the fusion protein plays an important role in increasing the solubility of the protein in addition to the important task of transferring the fusion protein into and out of the nucleus. When the fusion protein is located close to the C-terminal, It is more helpful to increase.

The term "in vivo stabilization protein" refers to a protein that, within a living body to which the fusion protein substantially acts, confers stability to allow the fusion protein to stably exist. The in vivo stabilizing protein should not induce immunogenicity in vivo as a part of the fusion protein, and in particular, any protein that can obtain stability in the blood of an object when administered to a subject is possible . According to one embodiment, the in vivo stabilizing protein is selected from the group consisting of AAT (alpha 1 antitrypsin), serum albumin, serum albumin binding peptide (SABP), Fc of immunoglobulin and polyethyleneglycol Or more, but is not limited thereto.

In one embodiment, when the in vivo stabilizing protein is introduced into the fusion protein, it may be included between the N-terminus, the C-terminus, or between the p53 protein and the p18 protein of the fusion protein.

The terminus of the fusion protein of the present invention may further include a stop codon. The termination codon can be inserted repeatedly two or more times, for example, the TAA sequence can be inserted repeatedly twice. In the present specification, the case where the stop codon is inserted two times repeatedly is referred to as STOPx2 for the sake of convenience.

The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, an annexin A2 binding protein, a p53 protein, or a p53 protein fragment having an amino acid sequence of SEQ ID NO: 1, and a fusion protein comprising a p18 protein do.

The fusion protein may further comprise at least one member selected from the group consisting of an in vitro stabilization protein, a membrane permeation sequence domain, a nuclear-cytoplasmic signal domain, and a stabilization protein in vivo, .

According to one embodiment, the fusion protein comprises a fusion protein comprising an annexin A2 binding protein, a p53 protein and a p18 protein in the order of N-terminal to C-terminal;

A fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of N-terminal to C-terminal;

a fusion protein comprising the p53 protein, the p18 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal; And

p18 protein, p53 protein, and annexin A2 binding protein in the order of N-terminal to C-terminal.

According to a preferred embodiment, the fusion protein is a fusion protein comprising an annexin A2 binding protein, a p53 protein and a p18 protein in the order of N-terminal to C-terminal, An outer stabilizing protein, a transmembrane sequence domain, and a nucleus-cytoplasmic signal domain, or may further comprise at least one selected from the group consisting of a transmembrane sequence domain in the C-terminal direction of the p18 protein, a nuclear-cytoplasmic signal domain, Wherein the fusion protein further comprises at least one member selected from the group consisting of a fusion protein and a stabilizing protein;

Terminal fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of the N-terminal to the C-terminal, wherein the in vitro stabilization protein, the transmembrane sequence domain and the nuclear- Signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain, and in vivo stabilization protein in the C-terminal direction of p53 protein A fusion protein further comprising;

a fusion protein comprising p53 protein, p18 protein, and annexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a membrane- Nuclear-cytoplasmic signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain and STOPx2 in the C-terminal direction of annexin A2 binding protein A fusion protein comprising at least a heterologous protein; And

a fusion protein comprising p18 protein, p53 protein and annexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a transmembrane sequence domain and / Nuclear-cytoplasmic signal domain, or in the group consisting of the transmembrane sequence domain, the nuclear-cytoplasmic signal domain and the in vivo stabilizing protein in the C-terminal direction of the annexin A2 binding protein And a fusion protein which further comprises at least one selected from the group consisting of a fusion protein and a fusion protein.

The present invention also provides a polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, and a cell transformed with the recombinant vector. The present invention also provides a method for producing a fusion protein comprising culturing the transformed cells.

The term "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form. The polynucleotide encompasses RNA genomic sequences, cDNA and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified. For example, the polynucleotide encoding the protein complex of the present invention may comprise the nucleic acid sequence of SEQ ID NO: 11.

The polynucleotide includes not only a nucleotide sequence coding for the amino acid sequence of the fusion protein but also a complementary sequence to the sequence. The complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences, which can be obtained under stringent conditions known in the art, for example, using nucleotides encoding the amino acid sequence of the fusion protein Quot; means a sequence that can hybridize with a nucleotide sequence of a nucleotide sequence.

The recombinant vector may be an expression vector capable of stably expressing the fusion protein in a host cell. The expression vector may be any conventional vector used in the art to express an exogenous protein in plants, animals or microorganisms. The recombinant vector may be constructed by a variety of methods known in the art.

The recombinant vector may be constructed with prokaryotic or eukaryotic cells as hosts. For example, when the vector is an expression vector and the prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, pL? Promoter, trp promoter, lac promoter, tac promoter, T7 promoter etc.) Lt; / RTI > sequence and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the origin of replication that functions in eukaryotic cells contained in the vector includes f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, and BBV replication origin But is not limited thereto. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV) can be used, and can generally include a polyadenylation sequence as a transcription termination sequence.

The host cells transformed in the present invention can be used to clone or express the recombinant vector in a stable manner. Any host cell known in the art can be used. Examples of prokaryotic cells include E. coli JM109, E. coli Bacillus subtilis, Bacillus subtilis, and Bacillus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Salmonella typhimurium, Yeast ( Saccharomyce cerevisiae ), insect cells, plant cells and animal cells such as a CHO cell line (for example, a yeast cell, a yeast cell, a yeast cell, etc.) Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines.

The delivery of the polynucleotide or a recombinant vector containing the polynucleotide into a host cell can be carried out by a method well known in the art. For example, when the host cell is a prokaryotic cell, the CaCl ₂ method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, Liposome-mediated transfection methods, and gene bombardment, but the present invention is not limited thereto.

The method of selecting the transformed host cells can be easily carried out according to a method widely known in the art by using a phenotype expressed by a selection marker. For example, when the selection mark is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

Culturing of the transformed cells can be carried out through various methods known in the art. For example, when the transformed cells are inoculated into a YT liquid medium and cultured, when IPTG is added to the medium at the time when the cell density reaches a certain level, protein expression by the lac Z promoter is induced and cultured, Lt; RTI ID = 0.0 > and / or < / RTI >

The protein secreted intracellularly or in the medium can be obtained in a purified form according to various purification methods known in the art. For example, by purification with solubility fractionation by ammonium sulfate, size fractional filtration and purification by various chromatographies (size, charge, hydrophobicity, or affinity-based separation) Proteins can be obtained. For example, when a fusion protein is fused to GST, a glutathione-coupled resin column is used. When the fusion protein is fused to 6x His, a desired protein can be easily obtained using a Ni ^{2 +} -NTA His-bonded resin column.

As described above, the fusion of p53 and p18 contained in the fusion protein greatly increases the stability of p53, transiently stops cell proliferation by expressing the p21 gene, induces cell death by expressing the Bax gene, 4, and 6, it is effective as an active ingredient in the prevention and / or treatment of cancer. In addition, the annexin A2 binding protein, another component contained in the fusion protein, binds to annexin A2, which is specifically expressed in cancer cells, so that cancer cells can be targeted so that the fusion protein selectively acts on cancer cells. Accordingly, the fusion protein of the present invention can be provided as a pharmaceutical composition for the prevention and / or treatment of cancer.

Therefore, in another example, there is provided a pharmaceutical composition for preventing and / or treating cancer comprising the fusion protein as an active ingredient. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent and / or excipient in an amount normally used.

The pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

In another embodiment, there is provided a method of preventing and / or treating cancer, comprising administering a therapeutically effective amount of the fusion protein to a patient in need of such prevention and / or treatment. The method for preventing and / or treating cancer may further comprise the step of identifying a patient in need of prevention and / or treatment of cancer before the administration step.

In yet another example, there is provided a use of the fusion protein for use in the prevention and / or treatment of cancer, or for use in the manufacture of a medicament for the prevention and / or treatment of cancer of the fusion protein.

The fusion protein or the pharmaceutical composition containing it as an active ingredient may be prepared in a unit dosage form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art, As shown in FIG. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

The pharmaceutical composition for preventing or treating the fusion protein or cancer comprising the fusion protein as an active ingredient may be orally or parenterally administered. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. In addition, the composition may be administered by any device capable of transferring the composition to a target cell.

The appropriate dosage of the pharmaceutical composition for preventing or treating the fusion protein or the cancer comprising the same as an effective ingredient can be determined depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration route, And response sensitivities. ≪ / RTI > The preferred dose of the composition is in the range of 0.001 to 100 mg / kg on an adult basis. The term " pharmaceutically effective amount "or" therapeutically effective amount "means an amount that is effective in preventing or treating cancer.

The subject to be administered with the pharmaceutical composition for preventing or treating the fusion protein or the cancer comprising the same as an active ingredient may be a mammal such as a rodent including a primate, a rat, a mouse and the like including a human, a monkey and the like.

According to one embodiment, the cancer to be prevented or treated by the fusion protein or pharmaceutical composition may be a solid cancer or a blood cancer, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, Endometrioid adenocarcinoma, adenocarcinoma, adenocarcinoma, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, gastrointestinal cancer, pancreatic cancer, endometrioid cancer, pancreatic cancer, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectum cancer, (Including a triple negative breast cancer), colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, mucin cancer, thyroid cancer, head and neck cancer, etc. But the present invention is not limited thereto.

The fusion protein according to one embodiment and the pharmaceutical composition comprising it as an effective ingredient can be effectively used for prevention or treatment of cancer. In addition, since the fusion protein is free of immunogenicity, it is useful as a safe anticancer therapy protein. It can be used for prevention and / or prevention of almost all kinds of cancer as well as having a specific effect by targeting only cancer cells, , And excellent effects can be expected also for solid cancer which does not exhibit the effect of existing anticancer drugs.

Fig. 1 schematically shows an example of a protein complex according to one embodiment.
Fig. 2 schematically shows the mechanism by which a protein complex according to one embodiment acts on cancer cells.
FIG. 3 shows the results of SDS-PAGE of the protein complexes # 1 and # 2 according to the present invention.
# 1: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -Ub (m3)
# 2: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -AAT-p53F-NLS
Figure 4 shows SDS-PAGE results confirming whether protein complexes # 1 and # 2 according to the present invention bind to annexin A2.
# 1: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -Ub (m3)
# 2: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -AAT-p53F-NLS
FIG. 3 shows the results of confirming cell growth inhibition effect according to the treatment concentration of protein complex # 2 in HCC1806 cell line.
# 2: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -AAT-p53F-NLS

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  1: Preparation of Expression Vector of Fusion Protein

In this embodiment, a ubiquitin wild-type protein or a ubiquitin mutant type protein, a membrane translocation sequence (MTS), p18, an alpha 1 antitrypsin (AAT), a p53 fragment, An expression vector of the protein complex for producing an intracellular delivery protein complex in the order of a nucleus localization signal domain (NLS) was prepared. For comparison with the protein complex, an expression vector for producing a protein complex excluding one or more components in the protein complex was prepared. Hereinafter, the mutant ubiquitin in which the wild-type ubiquitin is replaced with Ub7KR (SEQ ID NO: 5) and the wild-type ubiquitin C-terminal RGG is replaced with RGA is referred to as Ub7KR (G76A) (SEQ ID NO: 12). Ub (m1), Ub (m3), Ub (m4), and Ub (m4) were synthesized using the same amino acid sequence but using a wobble sequence. (SEQ ID NO: 13 to SEQ ID NO: 16), respectively.

Six types of expression vectors were purchased from Genentech, Inc., and pET-21b (+) (EMD Biosciences) was used as a vector for protein overexpression.

Each of the insert DNA fragments includes a nucleotide sequence that can be cleaved with NdeI at the 5'end and a nucleotide sequence that can be cleaved with XhoI at the 3'end so that the NdeI-XhoI cleavage of the pET21b (+) vector Lt; / RTI > sequence.

A schematic diagram illustrating a possible primary structure of a protein complex according to one embodiment is shown in FIG.

Example  2: Expression and purification of fusion protein

To over-express the protein complex using the vector prepared in Example 1, E. coli transformed with the vector BL21 (DE3). At this time, YT medium was used as a culture medium. When the OD value was 0.5 at an absorbance of 600 nm, 0.5 mM IPTG was added and further cultured at 18 ° C for 16 hours. The cultured cells were ultrasonically pulverized in 50 mM Tris-HCl buffer (pH 8.0) containing 5% glycerol, 5 mM? -Mercaptoethanol, 0.2% Triton X-100 and 0.2 M NaCl, Separator (10,000 g) was used to obtain the supernatant. The supernatant was applied to a Ni ^{2 +} -NTA superflow column (Qiagen) equilibrated with the buffer and eluted with a washing buffer (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM β- (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM [beta] -mercaptoethanol, 0.2% Triton X-100 And 0.2 M NaCl) to elute the protein complex. Protein complex-containing fractions were collected and the salts contained in the fractions were removed using an Amicon Ultra-15 Centrifugal Filter (Milipore) and concentrated. The purified protein concentration was measured using BSA as a standard.

FIG. 3 shows the result of purification of protein complexes expressed from the vector prepared in Example 1 by SDS-PAGE. In Figure 3, protein complex # 1 is a protein complex bound in the order of A2-Ub-Ub (m1) -p18-Ub (m3) -Ub (m4) (m1) -p18-AAT-p53. In order to confirm the binding with Annexin A2 protein, Annexin A2 was purified separately. M denotes a size marker .

Example  3: of the fusion protein Annexin  Confirmation of binding to A2

First, a binding test was conducted in vitro to determine whether the protein complex obtained in Example 2 actually binds to Annexin A2. First, A2-Ub-Ub (m1) -ub (m3) -Ub (m4) -p53, which is a protein complex # 1 comprising anexin A2 binding peptide, A2-Ub-Ub 500 μg of p18-AAT-p53 (# 2) and 1500 μg of annexin A2 protein were added to 1000 μl of PBS buffer (1 x PBS, 0.2% Tween 20, 100 mM arginine, 0.2% glutathione reduced) Min to provide sufficient binding time and then loaded onto a HiLoad Superdex S-75 16/60 column (GE healthcare) for elution. The elution conditions were as follows: flow rate 1 ml / min, PBS buffer (1 x PBS, 0.2% Tween 20, 100 mM arginine, 0.2% glutathione reduced) The eluate was then eluted with Ni ^{2 +} -NTA equilibrated with 50 mM Tris buffer (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM β-mercaptoethanol, 0.2% Triton X-100 and 1 M NaCl) superflow column (Qiagen), and the protein complex was eluted in the same manner as in Example 2. [

FIG. 4 shows SDS-PAGE of the protein complex eluted by the above procedure. As shown in FIG. 4, protein complexes # 1 and # 2, proteins containing anexin A2-binding peptide, were found to bind to annexin A2.

Example  4: Cancer cells  Identification of antitumor effect of fusion protein used

In order to confirm the therapeutic effect of intracellular permeation and cancer cells of the protein complex # 2 prepared in Example 2, triple negative breast cancer cell lines (HCC1806, ATCC) were cultured in the presence of p18-p53 The anticancer effect of the protein was confirmed.

The cells were each inoculated into RPMI medium (Gibco BL) containing 10% FBS so as to contain 5 × 10 ³ cells per well. The protein complexes were treated at concentrations of 0, 0.05625, 0.1125, 0.225, 0.45 and 0.9 μM, respectively , And incubated for 96 hours in a CO ₂ incubator at 37 ^° C, 85% humidity and 5% CO ₂ . As a comparative experiment, a protein-free buffer (0.1% arginine, 0.2% Tween 20, 0.2% L-Glutathione, 1XPBS (pH 7.4)) instead of the protein complex was treated in the same amount and cultured under the same conditions as above.

As a result, as shown in FIG. 5, the protein complex # 2 including both the nucleus locus signal domain (NLS) and the in vivo stable protein (AAT) inhibited cell growth of all the cell lines at a concentration of 0.9 uM by 50% .

<110> SAMSUNG ELECTRONICS CO., LTD. <120> Fusion protein comprising annexin A2 binding protein, p53 and          p18, and composition comprising the fusion protein for preventing          or treating cancer <130> DPP20125838 <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> p53 fragment <400> 1 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn   1 5 10 <210> 2 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> P18 protein <400> 2 Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly   1 5 10 15 Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn              20 25 30 Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly          35 40 45 Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp      50 55 60 Leu Lys Asp Arg Thr Gly Asn Ala Val Ile His Asp Ala Ala Arg Ala  65 70 75 80 Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val                  85 90 95 Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys             100 105 110 Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser         115 120 125 Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala     130 135 140 Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly 145 150 155 160 Ala Gly Aly Thr Asn Leu Gln                 165 <210> 3 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> p18-p53 fragment fusion protein <400> 3 Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly   1 5 10 15 Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn              20 25 30 Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly          35 40 45 Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp      50 55 60 Leu Lys Asp Arg Thr Gly Asn Ala Val Ile His Asp Ala Ala Arg Ala  65 70 75 80 Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val                  85 90 95 Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys             100 105 110 Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser         115 120 125 Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala     130 135 140 Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly 145 150 155 160 Ala Gly Aly Gly Aly Thr Asn Aly Gly Gly Gly Aly Gly Thr Phe Ser Asp Leu                 165 170 175 Trp Lys Leu Leu Pro Glu Asn             180 <210> 4 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Annexin A2 binding protein <400> 4 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys   1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Thr Gly Pro Gln              20 25 30 Cys Leu Cys Arg          35 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> wild type Ubiquitin <400> 5 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu   1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp              20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys          35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu      50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly  65 70 75 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> membrane transfer sequence (MTS) <400> 6 Ala Ala Val Ala Leu Ala Leu Pro Ala Val Leu Leu Ala Leu   1 5 10 15 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 7 Lys Lys Lys Arg Lys   1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 8 Pro Lys Lys Lys Arg Lys Val   1 5 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 9 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys   1 5 10 15 <210> 10 <211> 337 <212> PRT <213> Artificial Sequence <220> <223> AAT <400> 10 Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu   1 5 10 15 Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser              20 25 30 Pro Val Ser Ile Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys          35 40 45 Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr      50 55 60 Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg  65 70 75 80 Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly                  85 90 95 Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp             100 105 110 Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp         115 120 125 Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr     130 135 140 Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val 145 150 155 160 Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro                 165 170 175 Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val             180 185 190 Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile         195 200 205 Gln His Ala Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu     210 215 220 Gly Asn Ala Thr Ala Ile Phe Leu Pro Asp Glu Gly Lys Leu Gln 225 230 235 240 His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu                 245 250 255 Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile             260 265 270 Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr         275 280 285 Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala     290 295 300 Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp 305 310 315 320 Glu Lys Gly Thr Glu Ala Ala Pro Asp Asp Asp Asp Glu Ala Ile Pro                 325 330 335 Arg     <210> 11 <211> 2327 <212> DNA <213> Artificial Sequence <220> The polynucleotide encoding fusion protein (A2-Ub-Ub (m1) -p18-AAT-p53) <400> 11 catatgagag gatcgcatca ccatcaccat cacgattacg atatcccaac gaccgaaaac 60 ctgtattttc agggatccgg cagcggcagc atgtgcatgc cgtgcttcac caccgaccac 120 cagatggctc gtaaatgcga cgactgctgc ggtggtaaag gtcgtggtaa atgcaccggt 180 ccgcagtgcc tgtgccgtgg cagcatgcag atcttcgtta aaaccctgac cggtaaaacc 240 atcaccctgg aagttgaacc gtctgacacc atcgaaaacg ttaaagctaa aatccaggac 300 aaagaaggta tcccgccgga ccagcagcgt ctgatcttcg ctggtaaaca gctggaagac 360 ggtcgtaccc tgtctgacta caacatccag aaagaatcta ccctgcacct ggttctgcgt 420 ctgcgtggtg gtatgcaaat ctttgttcgt accctgaccg gtcgtaccat caccctggaa 480 gttgaaccgt cggataccat agaaaatgta cgtgcgcgta tccaggatcg tgaaggtata 540 ccgccggatc agcagcgtct gatctttgcg ggtcgtcagc tggaagatgg tcgcaccctg 600 tcggattaca atattcaacg tgaatcgacc ctgcatctgg tgctgcgtct gcgcggtgcg 660 gaatttgccg cggtagcgct gctcccggcg gtcctgctgg ccttgctggc gcccggcagc 720 atggccgagc cttgggggaa cgagttggcg tccgcagctg ccagggggga cctagagcaa 780 cttactagtt tgttgcaaaa taatgtaaac gtcaatgcac aaaatggatt tggaaggact 840 gcgctgcagg ttatgaaact tggaaatccc gagattgcca ggagactgct acttagaggt 900 gctaatcccg atttgaaaga ccgaactggt aatgctgtca ttcatgatgc ggccagagca 960 ggtttcctgg acactttaca gactttgctg gagtttcaag ctgatgttaa catcgaggat 1020 aatgaaggga acctgccctt gcacttggct gccaaagaag gccacctccg ggtggtggag 1080 ttcctggtga agcacacggc cagcaatgtg gggcatcgga accataaggg ggacaccgcc 1140 tgtgatttgg ccaggctcta tgggaggaat gaggttgtta gcctgatgca ggcaaacggg 1200 gctgggggag ccacaaatct tcaaggcagc accttcaaca aaatcacccc gaacctggct 1260 gaattcgctt tctctctgta ccgtcagctg gctcaccagt ctaactctac caacatcttc 1320 ttctctccgg tttctatcgc taccgctttc gctatgctgt ctctgggtac caaagctgac 1380 acccacgacg aaatcctgga aggtctgaac ttcaacctga ccgaaatccc ggaagctcag 1440 atccacgaag gtttccagga actgctgcgt accctgaacc agccggactc tcagctgcag 1500 ctgaccaccg gtaacggtct gttcctgtct gaaggtctga aactggttga caaattcctg 1560 gaagacgtta aaaaactgta ccactctgaa gctttcaccg ttaacttcgg tgacaccgaa 1620 gaagctaaaa aacagatcaa cgactacgtt gaaaaaggta cccagggtaa aatcgttgac 1680 ctggttaaag aactggaccg tgacaccgtt ttcgctctgg ttaactacat cttcttcaaa 1740 ggtaaatggg aacgtccgtt cgaagttaaa gacaccgaag aagaagactt ccacgttgac 1800 caggttacca ccgttaaagt tccgatgatg aaacgtctgg gtatgttcaa catccagcac 1860 gctaaaaaac tgtcttcttg ggttctgctg atgaaatacc tgggtaacgc taccgctatc 1920 ttcttcctgc cggacgaagg taaactgcag cacctggaaa acgaactgac ccacgacatc 1980 atcaccaaat tcctggaaaa cgaagaccgt cgttctgctt ctctgcacct gccgaaactg 2040 tctatcaccg gtacctacga cctgaaatct gttctgggtc agctgggtat caccaaagtt 2100 ttctctaacg gtgctgacct gtctggtgtt accgaagaag ctccgctgaa actgtctaaa 2160 gctgttcaca aagctgttct gaccatcgac gaaaaaggta ccgaagctgc tccggacgac 2220 gacgacgaag ctatcccgcg tgaaacattt tcagacctat ggaaactact tcctgaaaac 2280 aaaaagaaga gaaagtaata actcgagcac caccaccacc accactg 2327 <210> 12 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> mutant type Ubiquitin <400> 12 Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu   1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp              20 25 30 Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg          35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu      50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Ala  65 70 75 <210> 13 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m1) <400> 13 atgcaaatct ttgttcgtac cctgaccggt cgtaccatca ccctggaagt tgaaccgtcg 60 gataccatag aaaatgtacg tgcgcgtatc caggatcgtg aaggtatacc gccggatcag 120 cagcgtctga tctttgcggg tcgtcagctg gaagatggtc gcaccctgtc ggattacaat 180 attcaacgtg aatcgaccct gcatctggtg ctgcgtctgc gcggtgcg 228 <210> 14 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m2) <400> 14 atgcagattt tcgtgcgcac gctgacgggc cgcacgatca cgctggaggt tgagccgtcc 60 gacacgatag agaacgtacg cgcccgcatc caggaccgcg agggcatacc gccggaccag 120 cagcgcctga tctttgccgg ccgccagctg gaggacggcc gtacgctgtc cgactacaac 180 attcaacgcg agtccacgct gcatctggtg ctgagactgc gtggcgcc 228 <210> 15 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m3) <400> 15 atgcagatct tcgttcgcac cctgaccggc cgtacgatca cgctggaggt tgagccgtcc 60 gatacgatag agaatgtacg cgcgcgcatc caggatcgcg aaggcatacc gccggaccag 120 cagcgtctga tctttgcggg ccgtcagctg gaagacggtc gtaccctgtc ggactacaac 180 attcaacgtg agtcgacgct gcatctggtg ctgagactgc gtggtgcc 228 <210> 16 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m4) <400> 16 atgcaaattt ttgtgcgtac gctgacgggt cgcaccatca ccctggaagt tgaaccgtcg 60 gacaccatag aaaacgtacg tgcccgtatc caggaccgtg agggtatacc gccggatcag 120 cagcgcctga tctttgccgg tcgccagctg gaggatggcc gcacgctgtc cgattacaat 180 attcaacgcg aatccaccct gcatctggtg ctgcgtctgc gcggcgcg 228

Claims (15)

An annexin A2 binding protein, a p53 protein or a p53 protein fragment consisting of the amino acid sequence of SEQ ID NO: 1, and a p18 protein.
2. The method of claim 1, wherein the fusion protein comprises
An in vitro stabilization protein,
The membrane transfer sequence (MTS) domain,
A nucleus-cytoplasm signal domain, and
In vivo stabilization protein
&Lt; / RTI &gt; wherein the fusion polypeptide further comprises at least one polypeptide selected from the group consisting of:
3. The method according to claim 2, wherein the exo-stabilizing protein is at least one selected from the group consisting of wild-type ubiquitin, mutant ubiquitin, and ubiquitin-
The wild-type ubiquitin comprises the amino acid sequence of SEQ ID NO: 5,
Wherein the mutant ubiquitin is one in which at least one of the Lys present at positions 6, 11, 27, 29, 33, 48 and 63 of the amino acid sequence of the wild-type ubiquitin is substituted with Arg, Gly present at the 76th position of the wild- Ala, or at least one of Lys present at positions 6, 11, 27, 29, 33, 48 and 63 of the amino acid sequence of wild-type ubiquitin is substituted with Arg and Gly existing at the 76th position is substituted with Ala However,
Wherein said ubiquitin-like protein is selected from the group consisting of Nedd8, SUMO-1, SUMO-2, NUB1, PIC1, UBL3, UBL5 and ISG15.
Fusion protein.
3. The fusion protein of claim 2, wherein the transmembrane sequence domain is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7.
3. The fusion protein of claim 2, wherein the nuclear-cytoplasmic signal domain is a nucleus location sequence (NLS) domain or a nucleus export sequence (NES) domain.
6. The fusion protein according to claim 5, wherein the NLS domain is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:
The method according to claim 2, wherein the at least one member selected from the group consisting of the in vivo stabilizing protein AAT (alpha 1 antitrypsin), serum albumin, serum albumin binding peptide (SABP), Fc and PEG (polyethyleneglycol) , Fusion protein.
2. The method of claim 1, wherein the fusion protein comprises
A fusion protein comprising the annexin A2 binding protein, the p53 protein and the p18 protein in the order of N-terminal to C-terminal;
A fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of N-terminal to C-terminal;
a fusion protein comprising the p53 protein, the p18 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal; And
p18 protein, p53 protein and annexin A2 binding protein in the order of N-terminal to C-terminal
&Lt; / RTI &gt;
9. The method of claim 8, wherein the fusion protein comprises
Terminal fusion protein comprising the annexin A2 binding protein, the p53 protein and the p18 protein in the order of the N-terminal to the C-terminal in the N-terminal direction of the p53 protein, the in vitro stabilization protein, the transmembrane sequence domain, Signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain and in vivo stabilizing protein in the C-terminal direction of p18 protein A fusion protein further comprising;
Terminal fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of the N-terminal to the C-terminal, wherein the in vitro stabilization protein, the transmembrane sequence domain and the nuclear- Signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain, and in vivo stabilization protein in the C-terminal direction of p53 protein A fusion protein further comprising;
a fusion protein comprising p53 protein, p18 protein, and annexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a membrane- Nuclear-cytoplasmic signal domain, or in the group consisting of the transmembrane sequence domain, the nuclear-cytoplasmic signal domain and the in vivo stabilizing protein in the C-terminal direction of the annexin A2 binding protein A fusion protein which further comprises at least one selected; And
a fusion protein comprising p18 protein, p53 protein and anexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a transmembrane sequence domain and / Nuclear-cytoplasmic signal domain, or in the group consisting of the transmembrane sequence domain, the nuclear-cytoplasmic signal domain and the in vivo stabilizing protein in the C-terminal direction of the annexin A2 binding protein Wherein the fusion protein further comprises one or more selected &lt; RTI ID = 0.0 &gt;
&Lt; / RTI &gt;
10. A polynucleotide encoding the fusion protein of any one of claims 1 to 9.
10. A recombinant vector comprising the polynucleotide of claim 10.
A cell transformed with the recombinant vector of claim 11.
14. A method for producing the fusion protein of claim 1 comprising culturing the cell of claim 12.
9. A pharmaceutical composition for preventing or treating cancer comprising the fusion protein of any one of claims 1 to 9 as an active ingredient.
15. The method of claim 14, wherein the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, Small intestine cancer, soft tissue sarcoma, chronic or acute leukemia, lymphoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, Cancer of the endometrium or uterus, salivary gland cancer, kidney cancer, prostate cancer, mucin cancer, thyroid cancer, or head and neck cancer.

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