WO2014189335A1

WO2014189335A1 - Fusion protein comprising annexin a1-binding protein, p53 protein and p18 or p16 protein, and composition for preventing or treating cancer, comprising same

Info

Publication number: WO2014189335A1
Application number: PCT/KR2014/004644
Authority: WO
Inventors: 이정민; 이정훈; 김정민; 강혜윤; 이재일
Original assignee: 삼성전자 주식회사
Priority date: 2013-05-24
Filing date: 2014-05-23
Publication date: 2014-11-27
Also published as: KR20140138507A

Abstract

The present invention relates to a fusion protein, in which an annexin A1-binding protein, a p53 protein and a p18 or p16 protein are conjugated, the preparation of the fusion protein, and a pharmaceutical composition for preventing or treating cancer, comprising the fusion protein.

Description

【Specification】

[Name of invention]

Fusion protein comprising annexin A1 binding protein, p53 protein, and pl8 or pl6 protein and composition for preventing or treating cancer comprising the same

Technical Field

The present invention relates to an anneal A1 binding protein, a p53 protein, and a fusion protein to which a pl8 or pl6 protein is bound, preparation of the fusion protein, and a pharmaceutical composition for preventing or treating cancer comprising the fusion protein.

Background Art

To proliferate, cells cycle through the G1 phase, DNA replication phase, S2 phase, G2 phase, and M phase. This is called the cell cycle. . Cell division is a substantial step in which cells multiply through mitosis and cytoplasmic division. In a DNA replicator, DNA is ^' replicated and the number of cells remains unchanged. Therefore, in the cell cycle, DNA replicators, G1 and G2 groups, except for cell division, are called interphase. Groups G1 and G2 contain checkpoints that regulate the cell cycle, which plays an important role in preventing cells from proliferating abnormally. After checking at the checkpoint whether the conditions for passing to the next stage of the cell cycle have been met, the DNA replicator or cell divider will proceed if all conditions are met. Inadequate control of cell proliferation at these checkpoints leads to abnormal cell proliferation such as cancer.

To cleave past the G1 checkpoint, past the DNA replicator, and the G2 checkpoint, cyclin-dependent kinase (CDK) protein activity is required. CDK cannot be active alone, and it must be combined with Cyclin protein to have kinase activity. Cyclin-CD complex phosphorylates proteins involved in cell proliferation, resulting in cell division. Induce. Proteins that induce DNA replicators are mainly Cyclin D / E and CDK 4/6, and CDK 4/6 phosphorylates Rb (Retinoblastoma) protein to transcription of transcription factor E2F. Make it active. The proteins that induce cell division are mainly Cyclin A / B and CDK 2/1, and their Cyclin-CDK complex is called MPF (M-phase promoting factor). When mitosis or DNA replication is completed, no further kinase activity is required, which results in the degradation of Cyclin in the Cydin-CDK complex, which results in the loss of CDK activity.

There are many factors that regulate the activity of the Cyclin-CDK complex for cell cycle regulation. Exemplary variant is the p21 protein that act as all been ^'DNA damage checkpoint in G1. The p21 gene is expressed by p53, a tumor suppressor gene that is activated when DNA is damaged. p21 binds to the Cyclin-CDK complex that induces a DNA replicator and inhibits the kinase activity of CDK4 / 6/2, thereby preventing phosphorylation of the Rb protein. As a result, the cells stay in G1 phase and have time to repair damaged DNA. If the DNA damage is severely unacceptable to cells, p53 expresses the gene of Bax protein, an apoptosis-inducing gene. In other words, the action of p53 in the regulation of cell proliferation is to express p21 gene to temporarily stop cell proliferation, or p53 protein combined with ASPP protein, which is a p53-binding protein, expresses Bax gene. It can be divided into two things: apoptosis inducing. Apoptosis, also known as programmed cell death, is when cell death is beneficial at the individual level, such as when cells are no longer needed in the tissue, when they have been invaded by a virus or when they have been converted into cancer cells. It is a mechanism to commit suicide by itself.

CDK inhibitors that regulate cell cycle can be classified into two groups, 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 there are pl5, pl6, pl8 and pl9.

In order to regulate cell proliferation, a balance between the tumor suppressor gene and the proto-oncogene must be maintained. Mutation of the gene of the p53 protein, a tumor suppressor gene, leads to abnormal cell proliferation because it cannot induce apoptosis of abnormal cells. Also Precancerous genes, which are normal cell proliferation factors, are mutated (such as viral gene replacement) or overexpressed to become cancer genes, leading to abnormal cell proliferation such as cancer cells. The oncogene causes growth of cells with abnormal activity, intracellular signaling proteins, and thus causes false signal transduction, so that cell proliferation occurs even without external proliferation signals.

[Detailed Description of the Invention]

[Technical problem]

In view of this fact, the development of anticancer drugs using various factors that regulate the cell cycle in order to inhibit cancer caused by abnormal cell proliferation is required.

Technical Solution

One embodiment provides a fusion protein comprising an annexin A1 binding protein, a .p53 protein or fragment thereof, and a pl8 or pl6 protein.

Another embodiment provides a polynucleotide encoding the fusion protein.

Another embodiment provides a recombinant vector comprising the polynucleotide.

Another embodiment provides a cell transformed with the recombinant vector. Another embodiment provides a method of preparing 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 a subject a therapeutically effective amount of said fusion protein.

[Brief Description of Drawings]

1 schematically shows an example of a fusion protein according to one embodiment. 2 is a result showing that the fusion protein (protein complex # 1) binds to annexin A1 according to one embodiment, (1) is a result in the absence of calcium ions, (2) is a calcium ion The results are shown under these existing conditions.

Figure 3 shows the results confirming the cell growth inhibition effect according to the treatment concentration of the fusion protein (protein complex # 1) in HCC1806 cell line.

Figure 4 shows the results confirming the cell growth inhibition effect according to the treatment concentration of the fusion protein (protein complex # 1) in HCC116 cell line. [Best form for implementation of the invention]

One embodiment of the present invention provides a fusion protein comprising annexin A1 binding protein, p53 protein or fragment thereof, and pl8 or pl6 protein. The fusion protein may be an in vitro stabilization protein, a membrane transfer sequence (MTS) domain, a nuclear-cytoplasm signal domain, and an in vivo stabilization protein. It may further comprise one or more selected from the group consisting of.

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.

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 necessary.

As described above, CDK (Cyclin-dependent kinase) protein plays a major role in cancer development, and as an inhibitor thereof CIP (CDK inhibitor) protein lineage protein and INK4 (inhibitors of cyclin—dependent kinase 4) lineage proteins. The fusion protein according to one embodiment of the present invention is a p53 protein that regulates CIP strain protein expression and pl8 or pl6 protein belonging to the INK4 strain protein, and simultaneously exerts CIP strain protein activity and INK4 protein activity to improve cancer. Prophylactic and / or therapeutic effects may be obtained.

According to one embodiment, the p53 protein may be a polypeptide or fragment thereof having an amino acid sequence provided by NCBI accession number NPJD00537. P53 protein is used herein to mean both full-length protein and fragments thereof unless otherwise stated.

In addition, the p53 protein may be a transcriptional activation domain of p53 protein having the amino acid sequence of SEQ ID NO: KGlu 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 transcriptional active domain comprising the 18th to 26th amino acid sequence of the human p53 protein, which is a site that binds to Mdm2 in the p53 protein. The transcriptional activation domain of p53 significantly increases the stability of p53 in vivo by binding to Mdm2, which is responsible for p53 degradation, and expresses p21 gene encoding CDK inhibitor protein by p53 to temporarily stop cell proliferation. p53 combined with the apoptosis-stimulating protein of p53 (ASPP), a p53-binding protein, expresses the Bax gene and causes cell death. In addition, tumor suppressor pl8 or pl6 protein plays a role in inhibiting the activity of CDK 4, 6. Therefore, the fusion protein in which the transcriptional activation domain of p53 and the pl8 or pl6 protein are combined and the pharmaceutical composition containing the same as an active ingredient can be used as a novel anticancer agent.

According to one embodiment, the pl8 protein may be a polypeptide having an amino acid sequence of SEQ ID NO: 2. In addition, the pl8 protein may be used by modifying (substituting, deleting, adding, deleting, etc.) some amino acid residues for the purpose of improving solubility, so long as it does not affect the overall protein activity. According to one embodiment, the pl6 protein may be a polypeptide having an amino acid sequence of SEQ ID NO: 3. In addition, the pl6 protein may be used by modifying (substituting, deleting, adding, deleting, etc.) some amino acid residues so long as it does not affect the overall protein activity for the purpose of improving solubility.

The sequence of linking the p53 protein and pl8 or pl6 protein in the fusion protein is not limited, and includes both N-terminal -p53 protein -pl8 or pl6 protein -C terminus or N-terminal -pl8 or pl6 protein -p53 protein_C terminus forms. . In _one, embodiment, p53 protein fragments and pl8 protein binding fusion protein may be one having the amino acid sequence of SEQ ID NO: 4, p53 protein fragments and pl6 protein binding fusion protein having an amino acid sequence of SEQ ID NO: 5 It may be.

In addition, the fusion protein of the present invention includes the Annexin A1 binding protein (Annexin A1BP) ol. Annexin A1 binding protein binds to Annexin A1, which is specifically expressed in cancer cells, thereby enabling cancer cell targeting so that the fusion protein can selectively act only on cancer cells. Annexin A1 binding protein may be exemplified by a peptide having an amino acid sequence selected from SEQ ID NO: 6 to SEQ ID NO: 14, but the scope of the present invention is not limited thereto, as long as it can achieve cancer cell targeting activity Fragments, peptides, analogs or variants of the A1 binding protein may be used.

The annexin A1 binding protein in the fusion protein may be located at the N-terminus or C-terminus of the p53 and pl8 or pl6 fusions. For example, the fusion protein of the present invention may include an annexin A1 binding protein p53 protein, and a pl8 or ρΐβ protein in the order of Ν-terminal to C-terminal; A fusion protein comprising an annexin A1 binding protein, ρ18 or ρ16 protein, and ρ53 protein in the N-terminal order of the C-terminus; a fusion protein comprising a ρ53 protein, a ρ18 or ρ16 protein, and an annexin A1 binding protein in the order of Ν-terminal to C-terminal; a fusion protein comprising a ρ18 or ρ16 protein, a ρ53 protein and an annexin A1 binding protein in the order of Ν-terminal to C-terminal It may be.

In one preferred embodiment the fusion protein is an in vitro stabilizing protein

at least one member selected from the group consisting of in vitro stabilization protein, membrane transfer sequence (MTS) domain, nucleus-cytoplasm signal domain, and in vivo stabilization protein May include additional additions.

At this time, the position of the added polypeptide in the fusion protein is not limited, and the one or more polypeptides to be added may be included independently of the N-terminal, C-terminal, or p53 protein and pl8 or pl6 protein of the fusion protein. have.

Preferably, the fusion protein is stabilized in vitro in the N-terminal direction of the p53 protein in the fusion protein comprising an annexin A1 binding protein, p53 protein, and pl8 or pl6 protein in the N-terminal to C-terminal order Protein, membrane transmembrane sequence domain and nuclear-cytoplasmic signal domain, further comprising at least one selected from the group consisting of membrane permeable sequence domain, nuclear-cytoplasmic signal domain, and in vivo in the C-terminal direction of pl8 or pl6 protein. It may further comprise one or more selected from the group consisting of stabilizing proteins.

Also preferably, the fusion protein may include an annexin A1 binding protein, a pl8 or pl6 protein, and a p53 protein in the order of N-terminus to C-terminus, in the Ν—terminal direction of the pl8 or ρ16 protein. It further comprises one or more selected from the group consisting of an in vitro stabilizing protein, a membrane permeation sequence domain and a nuclear-cytoplasmic signal domain, or a membrane permeation sequence domain, a nuclear-cytoplasmic signal domain and a biomarker in the C—terminal direction of the ρ53 protein. It may further comprise one or more selected from the group consisting of a stabilizing protein.

Also preferably, the fusion protein may comprise a ρ53 protein, a ρ18 or ρ16 protein, and an annexin A1 binding protein, in the order of Ν-terminal to C-terminal, in the Ν-terminal direction of the annexin A1 binding protein. Further comprises at least one member selected from the group consisting of an in vitro stabilizing protein, a membrane permeation sequence domain and a nuclear-cytoplasmic signal domain, or is Annexin A1. The c-terminal direction of the binding protein may further include one or more selected from the group consisting of a membrane permeation sequence domain, a nuclear-cytoplasmic signal domain, and an in vivo stabilizing protein.

Also preferably, the fusion protein may comprise a pl8 or pl6 protein, a p53 protein, and an annexin A1 binding protein in the order of N-terminus to C-terminus. Direction further comprises at least one member selected from the group consisting of an in vitro stabilizing protein, a membrane permeation sequence domain and a nuclear-cytoplasmic signal domain, or a membrane permeation sequence domain, a nuclear- in the C-terminal direction of an annexin A1 binding protein. It may further comprise one or more selected from the group consisting of a cytoplasmic signal domain and an in vivo stabilizing protein.

As used herein, the term “in vitro stabilization protein” refers to a protein for enhancing the solubility and stability of the fusion protein outside of the fusion protein, that is, when the fusion protein is experimentally purified. Means. The ex vivo stabilizing protein is part of the fusion protein and should not induce immunogenicity in vivo. In one embodiment, when the ex vivo stabilizing protein is introduced into the fusion protein, it is preferably located in the N-terminal direction of the fusion protein, but is not limited thereto. Also preferably, the ex vivo stabilizing protein may additionally be located between pl8 and p53 in addition to both ends of the fusion protein.

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

Ubiquitin (Ub) is the most conserved protein found in nature and consists of 76 amino acid sequences and is a water soluble protein that shows perfect homology between evolutionarily diverse species such as insects, trout and humans. Ubiquitin is also known as a protein that is stable to changes in pH, is not easily denatured even at high temperatures, and is stable to proteases. Thus, ubiquitin can improve the insolubility of the fusion protein.

The ubiquitin or ubiquitin-like protein (Ubl) is wild type ubiquitin, wild type ubiquitin-like protein, mutant ubiquitin And mutant ubiquitin-like protein may be selected from the group consisting of. The mutant ubiquitin means that the amino acid sequence of the wild type ubiquitin is changed to another amino acid sequence, for example, ubiquitin in which Lys of wild type ubiquitin (SEQ ID NO: 15) is replaced with Arg, and / or wild type ubiquitin C ᅳ terminal RGG is RGA. Ubiquitin (ie, Gly present at 76th of the ubiquitin wild-type polypeptide is substituted with Ala). According to one embodiment, in the mutant ubiquitin in which Lys of the wild type ubiquitin is substituted with Arg, the substitution is at least one selected from Lys present in 6, 11, 27, 29, 33, 48 and 63 of the wild type ubiquitin. And substitutions may be made independently or in combination at the positions of Lys.

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

According to one embodiment, the ubiquitin or ubiquitin-like protein is

The C-terminus may comprise an amino acid sequence cleavable by the protease or an amino acid sequence not cleaved by the protease. Amino acid sequences cleavable by the protease can be identified through a search database known in the art. For example, a protease and a cleavable amino acid sequence thereof can be used which is searched for at http://www.expasy.org/tools/peptidecutter/peptidecutter—enzymes.html. When the cleavable amino acid sequence is included, the fusion protein is permeated into the cell, and then the ubiquitin or ubiquitin-like protein is cleaved by a protease in the cell, and includes annexin A1 binding protein, p53 protein, and pl8 or pl6 protein. The fusion protein is able to function in the cell. After the cleavage, the fusion protein may contain membrane transmembrane sequence domains and / or nuclear-cytoplasmic signal domains, but since these polypeptides are very short in length, they do not affect the function of the fusion protein. In addition, ubiquitin or Although ubiquitin-like proteins are not cleaved, ubiquitin or ubiquitin-like proteins are safe in vivo because they are not immunogenic, and because they do not fold because they do not contain cysteine, they do not cause fusion protein structural changes and the fusion proteins do not change in cells. It does not affect the function.

The term “membrane transfer” refers to the ability to transport a fusion protein to be delivered intracellularly and / or in vivo in vitro and / or in vivo.

In addition, "membrane transfer sequence (MTS) domain" means a polylapide having an amino acid sequence that can itself pass through the cell membrane of a phospholipid bilayer. 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). Characterized in having a. Thus, the physical properties of the membrane permeable sequence domains usually exhibit hydrophobicity. According to one embodiment, the membrane permeable sequence domain may be any polypeptide having an amino acid sequence that can pass through the cell membrane of the phospholipid bilayer per se and is not particularly limited, but may be composed of the amino acid sequence of SEQ ID NO: 16 .

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

The term "nucleus-cytoplasm signal domain" is interpreted to mean a polydide sequence that serves to transport fusion proteins into or out of the nucleus. According to one embodiment, the nuclear cell cytoplasmic signal domain may be a NLSCnucleus location sequence (NLSCnucleus location sequence) domain or NES (nucleus export sequence) domain. That is, NLS may be included in the fusion protein to transfer the fusion protein into the nucleus, and NES may be included in the fusion protein in order to keep the fusion protein in the cytoplasm. NLS domains are proteins that are transported from the cytoplasm to the nucleus, and NES domains are characterized by proteins that are transported from the nucleus to the cytoplasm, all of which refer to polypeptides having an amino acid sequence that can pass through the nuclear membrane. The polypeptide which may be used as the NLS domain is not particularly limited. For example, the polypeptide may be KKKRK (SEQ ID NO: 17), PKKKRKV (SEQ ID NO: 18), KRPAATKKAGQAKKKK (SEQ ID NO: 19), 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 moving the fusion protein into and out of the nucleus, where it is located close to the C-terminus of the fusion protein. It is more helpful for the increase.

The term “in vivo stabilization protein” refers to a protein that provides stability to the fusion protein stably in a living body in which the fusion protein substantially acts. The protein is a part of the fusion protein, and should not induce immunogenicity in vivo, and in particular, any protein can be used as long as it can obtain stability in the blood of the subject. According to the present invention, the in vivo stabilizing protein may be at least one selected from the group consisting of AAT (alpha 1 antitrypsin), serum albumin, serum albumin binding peptide (SABP), immunoglobulin Fc and PEG (polyethyleneglycol). In one embodiment, the in vivo stabilizing protein is fused. When incorporated into the protein, it may also be implemented between the N-terminal, C-terminal or the p53 protein and pl8 protein of the fusion protein.

Also, of the present invention _. At the end of the fusion protein, a stop codon may be further included. The stop codon may be inserted repeatedly two or more times, for example, a TAA sequence may be inserted twice. In the present specification, the case where the stop codon is inserted twice is referred to as STOPx2 for convenience. In addition, the present invention provides an annexin A1 binding protein, p53 protein or p53 protein fragment having the amino acid sequence of SEQ ID NO: 1, and pl8 or pl6 protein It provides a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein comprising as an active ingredient.

The fusion protein may further include one or more selected from the group consisting of an in vitro stabilizing protein, a membrane permeation sequence domain, a nuclear-cytoplasmic signal domain, and an in vivo stabilizing protein, and the details thereof are as described above. .

According to one embodiment, the fusion protein is a fusion protein comprising an annexin A1 binding protein, p53 protein, and ρ18 or pl6 protein in the N-terminal to C-terminal order;

A fusion protein comprising an annexin A1 binding protein, a pl8 or pl6 protein, and a p53 protein in the order of the N-terminus to the C-terminus;

a fusion protein comprising a p53 protein, a pl8 or pl6 protein, and an annexin A1 binding protein in the order of N-terminus to C-terminus; And

The pl8 or pl6 protein, p53 protein and annexin A1 binding protein may be selected from the group consisting of a fusion protein comprising in the order of the N- terminal to the C- terminal.

According to another preferred embodiment, the fusion protein is an annexin A1 binding protein, p53 protein, and pl8 or pl6 protein in the fusion protein comprising N-terminal Ο terminal, N-terminal direction of the p53 protein Or at least one member selected from the group consisting of an ex vivo stabilizing protein, a membrane permeation sequence domain and a nuclear cytoplasmic signal domain, or a membrane permeation sequence domain, a nuclear cytoplasmic signal in the C-terminal direction of a pl8 or pl6 protein. A fusion protein further comprising at least one selected from the group consisting of a domain and an in vivo stabilizing protein; ，

In fusion proteins comprising annexin A1 binding protein, pl8 or pl6 protein, and p53 protein in the N-terminus to C-terminus, in vitro stabilizing protein in the N-terminal direction of pl8 or pl6 protein, membrane permeation sequence Further comprising one or more selected from the group consisting of a domain and a nuclear cytoplasmic signal domain, or selected from the group consisting of a membrane permeable sequence domain, a nuclear-cytoplasmic signal domain and an in vivo stabilizing protein in the C-terminal direction of the P53 protein. 1 kind A fusion protein further comprising an ideal phase;

In a fusion protein comprising p53 protein, pl8 or pl6 protein, and annexin A1 binding protein in the order of N-terminus to C-terminus, in vitro stabilizing protein, membrane permeation in the N-terminal direction of annexin A1 binding protein, It further comprises one or more selected from the group consisting of a sequence domain and a nuclear-cytoplasmic signal domain, or a membrane permeable sequence domain, a nuclear ᅳ cytoplasmic signal domain and an in vivo stabilizing protein in the C-terminal direction of the annexin A1 binding protein. A fusion protein further comprising one or more selected from the group consisting of; And

In a fusion protein comprising pl8 or pl6 protein, p53 protein and annexin A1 binding protein in the order of N-terminus to C-terminus, in vitro stabilizing protein, membrane permeation sequence in the N-terminal direction of annexin A1 binding protein Or at least one member selected from the group consisting of a domain and a nuclear cell cytoplasmic signal domain, or a membrane permeable sequence domain, a nuclear cytoplasmic signal domain, and an in vivo stabilizing protein in the C-terminal direction of the annexin A1 binding protein. It may be selected from the group consisting of a fusion protein that further comprises one or more selected from the group.

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. In addition, the present invention provides a method for producing a fusion protein comprising the step of culturing the transformed cells.

The term "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides present in single- or double-stranded form. Such polynucleotides encompass RNA genomic sequences, cDNAs and RNA sequences transcribed therefrom, and include analogs of natural polynucleotides unless specifically noted otherwise. For example, the polynucleotide encoding the protein complex of the present invention may be one comprising the nucleic acid sequence of SEQ ID NO: 21.

The polynucleotide includes not only the nucleotide sequence encoding the amino acid sequence of the fusion protein but also a complementary sequence to the sequence. The complementary sequence is perfectly In addition to complementary sequences, it also includes substantially complementary sequences that can be hybridized with nucleotide sequences encoding, for example, the amino acid sequence of the fusion protein, under stringent conditions known in the art. Means.

The recombinant vector may be an expression vector, which can stably express the fusion protein in a host cell. The expression vector may be a conventional one used to express foreign proteins in plants, animals or microorganisms in the art. The recombinant vector can be constructed through various methods known in the art.

The recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, if the vector is an expression vector and the prokaryotic cell is the host, a strong promoter capable of promoting transcription (e.g., pLA promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), translation It is common to include ribosomal binding sites and transcription / detox termination sequences for the initiation of. In the case of eukaryotic cells as hosts, replication origins that operate in eukaryotic cells included in the vector include fl replication origin, SV40 replication origin, pMBl replication origin, adeno replication origin, AAV replication origin, and BBV replication origin. It is not limited. Also, promoters derived from the genome of mammalian cells (eg, metallothionine promoters) or promoters derived from mammalian viruses (eg, adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus promoter and tk promoter of HSV) may be used and may generally include a polyadenylation sequence as a transcription termination sequence.

In the present invention, the transformed cell may be any host cell known in the art as the host cell capable of continuously cloning or expressing the recombinant vector, and as a prokaryotic cell, for example, E. coli Bacillus genus strains such as JM109, E. coll BL21, E. coli RRl, E. coJi LE392, E. coli B, E. coli X 1776, E. co // W3110, Bacillus subtilis, Bacillus thuringiensis, And enterococci and strains such as Salmonella typhimurium, Serratia marsonsons and various Pseudomonas species. In the case of conversion, as a host cell, Ho—SX Saccharomyce cerevisiae, stromal cells, plant cells and animal cells, for example, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN And MDCK cell lines can be used.

The delivery of the polynucleotide or recombinant vector comprising the same into a host cell may employ a delivery method well known in the art. For example, when the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method may be used. When the host cell is a eukaryotic cell, a micro-injection method, a calcium phosphate precipitation method, an electroporation method, a liposome -Mediated transfection and gene bombardment may be used, but is not limited thereto.

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

Cultivation of the transformed cells can be carried out through various methods known in the art. For example, the cells were cultured by inoculating the transformed cells in YT liquid medium, and then inducing and culturing protein expression by the / acZ promoter by adding IPTG to the medium when the cell density reached a certain level. Proteins secreted in vitro or in media can be obtained.

Proteins secreted into cells or into the medium can be obtained in purified form according to various purification methods known in the art. In purified form, for example, by solubility fractionation with ammonium sulphate, size fractional filtration and purification by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). Protein can be obtained. For example, when the fusion protein is fused to GST, the desired protein can be easily obtained by using a resin column bound to glutathione or a Ni ²⁺ -NTA His-bound resin column when fused to 6x His.

As described above, p53 and pl8 included in the fusion protein Or the pl6 fusion significantly increases the stability of p53, temporarily expresses the p21 gene and stops cell proliferation, and expresses the Bax gene to induce cell death and inhibit CDK 4, 6 activity. It is effective as an active ingredient in the prevention and / or treatment of cancer. In addition, annexin A1 binding protein, another component included in the fusion protein, binds to annexin A1 specifically expressed in cancer cells, thereby enabling cancer cell targeting so that the fusion protein can selectively act on only cancer cells. Therefore, the fusion protein of the present invention can be provided as a pharmaceutical composition for the prevention and / or treatment of cancer.

Thus, in another embodiment, 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 as necessary.

The pharmaceutically acceptable carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stearic acid magnesium and mineral oils, including but not limited to no. The pharmaceutical composition may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspension preservative, and the like, in addition to the above components.

In another example, a method of preventing and / or treating cancer is provided, comprising administering a therapeutically effective amount of the fusion protein to an individual in need thereof. The method for preventing and / or treating cancer may further comprise identifying a subject in need of ^' prophylaxis and / or treatment of cancer prior to the administering step.

In another example, use is provided for use in the prevention and / or treatment of cancer of the fusion protein, 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 a pharmaceutical composition comprising the same as an active ingredient is a pharmaceutically acceptable carrier according to a method which can be easily carried out by those skilled in the art. And / or by formulating with excipients, they may be prepared in unit dose form or may be prepared within 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 accelerators, and may further comprise dispersants or stabilizers. In addition, the composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional medical agents.

The fusion protein or a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration. In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device that allows the composition to migrate to the target cell.

Suitable dosages of the fusion protein or a pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient include a formulation method, a mode of administration, a patient's age, weight, sex, morbidity, food, time of administration, route of administration, and excretion rate. And various factors such as reaction responsiveness. Preferred dosages of the compositions are 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 can be effective in preventing or treating cancer.

The patient to be administered the pharmaceutical composition for preventing or treating the fusion protein or cancer containing the same as an active ingredient may be a rodent including a mammal, for example, a primate including a human monkey, and the like.

According to one embodiment, the cancer to be prevented or treated by the fusion protein or pharmaceutical composition may be solid or hematologic cancer, for example, 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 myeloma, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or Acute leukemia, lymphoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulva cancer, thyroid cancer, head and neck cancer It may be selected from the group struggled by the back, but is not limited thereto.

[Form for implementation of invention]

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples. Example 1 Preparation of Expression Vectors of Fusion Proteins

In this embodiment, one of the hydrophilic polypeptides is ubiquitin wild type protein or ubiquitin mutant protein, membrane transfer sequence (MTS), pi 8, in vivo stabilizing protein (AAT: alpha 1 antitrypsin), p53 fragment, nucleus. An expression vector of the protein complex was prepared to produce a protein complex for intracellular delivery linked in the order of the nucleus localization signal domain (NLS). In addition, an expression vector for producing a protein complex excluding one or more components from the protein complex was prepared for a comparative experiment with the protein complex. Hereinafter, wild type ubiquitin was referred to as Ub, and a wild type ubiquitin C-terminal RGG was prepared. The mutant ubiquitin modified with RGA is called Ubml.

A total of two kinds of expression vectors were prepared by Genotech Co., Ltd., and a vector for protein overexpression was pET-21b (+) (EMD Biosciences).

On the other hand, each insert DNA fragment comprises a nucleotide sequence that can be cleaved with Ndel at the 5 'end, and a nucleotide sequence that can be cleaved with Xhol at the 3' end, Ndel-Xhol cleavage of the pET21b (+) vector Can be inserted into the sequence. A schematic diagram showing possible primary structures of the protein complex according to one embodiment is shown in FIG. 1. Example 2: Expression and Purification of Fusion Proteins

In order to overexpress protein complex # 1 (Fig. 1 (a), SEQ ID NO: 21) using the vector prepared in Example 1, it was expressed in E. coli BL2 KDE3 transformed into the vector. . At this time, YT medium was used as a culture medium, and when the OD value was 0.5 at an absorbance of 600 nm, 0.5 mM IPTG was added thereto and further incubated at 18 ^° C. for 16 hours. Cells obtained by the culture were pulverized by ultrasonication in 50 mM Tris-HCl complete solution (pH 8.0) containing 5% glycerol, 5 mM β-mermer ethanol, 0.2% Triton X-100 and 0.2 M NaCl. The supernatant was obtained using a centrifuge (10,000 g). The supernatant was applied to a Ni ²⁺ -NTA superflow column (Qiagen) equilibrated with the complete solution, and the washed supernatant (50 mM Tris-HCl, pH 8.0, 5% glycerol) corresponding to 5 times the column volume. Wash the 5 mM β-mercaptoethane with 0.2% Triton X-100 and 1 M NaCl, and then elute the complete solution (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM β-mercapto). Ethane was eluted with 0.2% Triton X-100 and 0.2 M NaCl). Fractions containing the protein complex were collected and the salt contained in the fraction was removed and concentrated using Amicon Ultra-15 Centrifugal Filter (MUipore). Purified protein concentration was measured using BSA as a standard. Example 3: Confirmation of Calcium-dependent Binding of the Fusion Protein to Annexin A1 The protein complex # 1 and annexin A1 prepared in Example 2 contained calcium-free buffer (Tris-HCl 50 mM, 10% glycerol pH 7.6) or calcium. After incubation with a buffer (Tris-HCl 50mM, 10% glycerol pH 7.6, CaC12 5mM), size exclusion chromatography was performed using a Superdex200 column. The protein solution passed through the column was obtained by fraction and confirmed by SDS-PAGE electrophoresis.

As a result, as shown in FIG. 2, the protein complex and annexin A1 were elution in different fractions when there was no calcium, whereas the protein complex and annexin A1 were elution in the same fraction in the presence of calcium. . This shows that the protein complex and annexin A1 bind in the presence of calcium, and that the protein complex for annexin A1 targeting stratification is Annexin Al binding force could be confirmed. Example 4: Confirmation of anticancer effect of fusion protein using cancer cell line

To confirm the intracellular permeation and therapeutic efficacy of cancer cells of the protein complex # 1 prepared in Example 2, the protein complex of the triple negative breast cancer cell line (HCC1806, ATCC) and colon cancer cell line (HCT116, ATCC) The anticancer effect of pl8-p53 protein by administration was confirmed.

The cells were each dispensed in RPMI medium (Gibco BL) containing 10% FBS with 5><10 ³ per well in a 96-well plate. The next day, the proteins were 0, 0.25, 0.5, 1, 2, 4uM and then incubated for 72 hours at a temperature of 37 ⁰ C, C0 ₂ 5% in a CO ₂ incubator. In the comparative experiment, the protein complex-free buffer (0.1 arginine, 0.2% Tween20, 0.2% L-Glutathione, lXPBSCpH 7.4) was treated in the same amount and cultured under the same conditions.

As a result, as shown in Figures 3 and 4, it was confirmed that the protein complex inhibits the cell growth of more than 50% in both HCC1806, HCT116 cells at the concentration of l ~ 2uM.

Claims

[Range of request]

[Claim 1]

An fusion protein comprising an annexin A1 binding protein, a p53 protein or a p53 protein fragment consisting of the amino acid sequence of SEQ ID NO: 1, and a pl8 or pl6 protein.

[Claim 2]

The method of claim 1, wherein the fusion protein

In vitro stabilization protein, ^' membrane transfer sequence (MTS) domain, nucleus-cytoplasm signal domain, and

In vivo stabilization protein

The fusion protein further comprises one or more polypeptides selected from the group consisting of.

[Claim 3]

According to claim 2, wherein the ex vivo stabilizing protein is at least one selected from the group consisting of wild type ubiquitin, mutant ubiquitin, and ubiquitin-like protein,

The wild type ubiquitin is composed of the amino acid sequence of SEQ ID NO: 15,

The mutant ubiquitin is one wherein at least one of Lys in 6, 11, 27, 29, 33, 48 and 63 of the amino acid sequence of the wild type ubiquitin is replaced with Arg, and the Gly present in the 76th of the wild type ubiquitin is with Ala ^'it is substituted, or a Gly to the wild-type one or more of ubiquitin amino acids 6, 11, 27, 29, Lys present in 33, 48 and 63-th sequence is substituted by Arg present in the 76th to Ala Is substituted,

The ubiquitin-like protein is selected from the group consisting of Nedd8, SUM 1, SUMO-2, NUBl, PIC1, UBL3, UBL5 and ISG15, fusion protein.

[Claim 4]

The fusion protein of claim 2, wherein the membrane permeable sequence domain is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16. 4.

[Claim 5]

The fusion protein of claim 2, wherein the nuclear-cytoplasmic signaling domain is a nucleus location sequence (NLS) domain or a NESCnucieus export sequence (NESC) domain.

[Claim 6]

The fusion protein of claim 5, wherein the NLS domain is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. 7.

[Claim 7]

According to claim 12, In vivo stabilizing protein AAT (alpha 1 antitrypsin), serum albumin, serum albumin binding peptide (serum albumin binding peptide (SABP), Fc and PEG (polyethyleneglycol) is one or more selected from the group consisting of , Fusion protein.

[Claim 8]

The method of claim 1, wherein the fusion protein

A fusion protein comprising an annexin A1 binding protein, a p53 protein, and a pl8 or pl6 protein in the N-terminus to the C-terminus;

pl8 or pl6 protein, p53 protein and annexin A1 binding protein N- Fusion protein comprising c-terminus at the terminal

The fusion protein, which is selected from the group consisting of.

[Claim 9]

The method of claim 8, wherein the fusion protein is

A fusion protein comprising an annexin A1 binding protein, a p53 protein, and a pl8 or pl6 protein in the N-terminus to C-terminus order, comprising: an in vitro stabilizing protein, a membrane permeable sequence domain in the N-terminal direction of the p53 protein, and nucleus-incorporated by more than one kinds of all ^'is selected from the group consisting of the cytoplasmic domain, or signal, or pl6 pl8 transmembrane to C- terminal direction of the protein sequence, domain, nuclear-from the group consisting of the cytoplasmic domain signal and in vivo stabilization protein A fusion protein further comprising at least one selected;

In a fusion protein comprising an annexin A1 binding protein, a pl8 or pl6 protein, and a p53 protein in the N-terminus to the C-terminus, an in vitro stabilizing protein in the N 투과 terminal direction of the pl8 or pl6 protein, a membrane permeation sequence At least one selected from the group consisting of a domain and a nuclear-cytoplasmic signal domain, or selected from the group consisting of a membrane permeable sequence domain, a nuclear-cytoplasmic signal domain and an in vivo stabilizing protein in the C-terminal direction of the p53 protein. A fusion protein further comprising one or more;

In a fusion protein comprising p53 protein, pl8 or pl6 protein, and annexin A1 binding protein in the N-terminus to C-terminus order, the ex vivo stabilizing protein membrane permeation sequence in the N-terminal direction of the annexin A1 binding protein. At least one selected from the group consisting of a domain and a nuclear-cytoplasmic signal domain, or a membrane permeable sequence domain, a nuclear-cytoplasmic signal domain, and an in vivo stabilizing protein in the C-terminal direction of the annexin A1 binding protein. A fusion protein further comprising at least one selected from the group; And

In a fusion protein comprising pl8 or pl6 protein, p53 protein and annexin A1 binding protein in the order of N-terminus to C-terminus, in vitro stabilizing protein, membrane permeation sequence in the N-terminal direction of annexin A1 binding protein At least one member selected from the group consisting of a domain and a nuclear-cytoplasmic signaling domain. A fusion protein further comprising or further comprising a subtype selected from the group consisting of a membrane permeable sequence domain, a nuclear-cytoplasmic signal domain, and an in vivo stabilizing protein in the C-terminal direction of the annexin A1 binding protein.

The fusion protein, which is selected from the group consisting of.

[Claim 10]

A polynucleotide encoding the fusion protein of any one of claims 1 to 9.

[Claim 11]

A recombinant vector comprising the polynucleotide of claim 10.

[Claim 12]

Cells transformed with the recombinant vector of claim 11

[Claim 13]

A method of making the fusion protein of claim 1, comprising culturing the cells of claim 12. [Claim 14]

A pharmaceutical composition for preventing or treating cancer, comprising the fusion protein of any one of claims 1 to 9 as an active ingredient.

[Claim 151]

15. The method of claim 14, wherein the cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular myeloma, rectal cancer, anal muscle cancer, esophageal cancer, Small bowel cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colon cancer, uterus Endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, or A pharmaceutical composition for preventing or treating cancer, which is head and neck cancer.

[Claim 161

The fusion protein of any one of claims U to C.

A method of treating cancer, comprising administering to a subject.