KR101332621B1 - Biopin - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a biopin, and is a fusion protein in which a cell penetration protein and a cohesive peptide are combined around a cell membrane protein, and are used for cell delivery and treatment, cell tracking, targeting of specific tissues and cells, molecular imaging systems, and medical care. It can be used for tools.

Description

Biopin

The present invention is a fusion protein in which a cell penetration peptide and a cohesive peptide are combined around a cell membrane protein, and can be used for cell delivery and treatment, tracking of cells, targeting of specific tissues and cells, molecular imaging systems, and medical tools. Relates to sex bioffin.

Recent stem cell transplantation, tissue transplantation, nanotubes, nanowires, microelectromechanical systems (MEMS), nanoparticles, biosensors, biochips , Development of biomaterials capable of controlling adhesion in fields such as drug delivery (Nahar et al.) al ., 2006, Crit Rev Ther Drug Carrier Syst , 23, 259-318). However, stem cell delivery, for example, delivers possible endothelial progenitor cells (EPCs) to myocardial tissue as delivery cells for the treatment of myocardial infarction. Attached to the tissue, making it very inefficient (Aicher et al ., 2003, Circulation , 107, 2134-2139). Most other delivery cells have similar results, and various attempts have been made to overcome them, but no breakthroughs are available. In particular, researches to improve cell attachment effects by delivering genes of adhesion protein to cells have been conducted (Sheyn et. al ., 2010, Adv Drug Deliv Rev. , 62, 683-698), overexpressed cells are genetically modified cells that are likely to be regulated in future clinical applications, making them difficult to commercialize.

Recently, researches on developing bioadhesives using biocompatible materials have been conducted in various fields. Typically, the adhesive, Naturalock's urethane adhesive (urethane-based adhesive foam), starch-based adhesive (starch-based adhesives) obtained from soybean, blue mussel (blue mussel. Mytilus Edulis ) -derived adhesives and mussel-derived protein adhesives have been developed for industrial and medical applications (Ciannamea et al ., 2010, Bioresour). Technol , 101, 818-825; Luhrs and Geurtsen, 2009, Prog Mol Subcell Biol , 47, 359-380; Valenta, 2005, Adv Drug Deliv Rev , 57, 1692-1712; Silverman and Roberto, 2007, Mar Biotechnol , 9, 661-681). However, these bioadhesives are environmentally friendly compared to other polymer-based adhesives such as epoxy and phenolic resins, but their primary purpose is to develop more powerful materials for surgical or industrial bonding applications. Not suitable for direct application.

SUMMARY OF THE INVENTION An object of the present invention is to provide a peptide biopin that can increase physical adhesion between cells by using cell adhesion peptides, a method for preparing the same, and a use thereof as a nano biomaterial such as cell delivery, cell therapy, diagnosis and treatment system. .

In order to achieve the above object, the present invention is a cell membrane protein;

One or more cell-penetrating peptides (CPPs) bound to the protein; And

Provided is a fusion protein comprising one or more cell adhesive peptides bound to the protein.

The invention also provides a recombinant vector expressing a fusion protein according to the invention.

The invention also provides a transformant transduced with the recombinant vector according to the invention.

The present invention also provides a method for producing a fusion protein of the present invention comprising the step of separating and purifying the fusion protein from the culture of the transformant according to the present invention.

The invention also relates to cell membrane proteins;

One or more cell-penetrating peptides (CPPs) bound to the protein; And

One or more cell adhesive peptides bound to the protein;

The cell penetrating peptide provides a fusion protein to which a fluorescent material is bound.

According to one embodiment, the cell-penetrating peptide is characterized in that the tissue-specific binding component or pharmaceutically active ingredient is further bound.

The present invention also provides a fusion protein according to the present invention to which the fluorescent material is bound; And

Provided is a contrast agent composition comprising a pharmaceutically acceptable carrier.

The present invention also relates to a fusion protein according to the present invention to which a fluorescent substance and a tissue specific binding component are bound; And

Provided is a target-oriented contrast agent composition comprising a pharmaceutically acceptable carrier.

The present invention also provides a fusion protein conjugated with a fluorescent substance and a pharmaceutically active ingredient; And

It provides a diagnostic or therapeutic contrast composition comprising a pharmaceutically acceptable carrier.

The present invention also relates to a fusion protein according to the present invention to which a fluorescent substance is bound; And

Provided are multiple diagnostic probes, including diagnostic probes.

The invention also relates to fusion proteins according to the invention; And

It provides a cell therapy comprising at least one stem cell or immune cell.

The invention also relates to fusion proteins according to the invention; And

Provided is a medical stent comprising at least one endothelial cell or vascular progenitor cell.

The fusion protein of the present invention is a biopin that is effectively delivered to cells and increases physical binding between cells, and is applicable to drugs or cell delivery, cell therapy, molecular imaging, medical tools, and the like for diagnosis or treatment. It can be usefully used as.

1 is a fusion protein of the present invention prepared by fusing an adhesive peptide containing RGD and a cell-penetrating peptide, respectively, at the N-terminus and C-terminus of a cell membrane protein (FIG. 1). The figure shows a fusion protein (bottom figure) in which cell-penetrating peptides are fused at the C-terminus with respect to cell membrane proteins, thereby preventing intercellular binding.
Figure 2 shows the nucleotide sequence and amino acid sequence of the fusion protein (BPin-RGD) according to an embodiment of the present invention, the second line of the box is a sequence containing the adhesive peptide RGD, the fourth box of the box Is TAT-CPP, a type of cell penetrating peptide, and the sequence between the boxes is part of opioid receptor delta transmembrane domain 1 (OPRD) of the opioid receptor delta.
Figure 3 shows the structure of the fusion protein of the present invention, the left blue italic body of BPin-RGD is a sequence containing the adhesive peptide RGD, the right italic body is a cell penetration peptide and the underline is the transmembrane domain 1 of the opioid receptor delta BPin-44 is a form of a cell penetrating peptide coupled to the C-terminus.
4 is a confocal microscope photograph obtained by treating cells with the fusion proteins (BPin-44 and BPin-RGD) of the present invention.
Figure 5 is treated with cardiac cells H9c2 treated with the fusion protein (BPin-RGD) of the present invention to rat mesenchymal stem cells (MSCs) of rats stained with Green fluorescent dye (DiO) ) Is a graph obtained by calculating the absorbance / emission (484 nm / 501 nm) value measured by a fluorescent spectrometer by adhering to a petri dish.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention aims to develop a biopin that can increase the physical binding between cells rather than a method of overexpressing a specific gene that is difficult to predict biological changes in cells and has side effects.

In general, therapeutic effects may vary depending on how effectively the delivered cells initially attach to target organs and tissues in cell delivery and cell therapy techniques. Various attempts to efficiently deliver delivery cells to target tissues using methods of overexpressing specific genes or polymeric compounds are disproving. Therefore, unlike the conventional genetic engineering method or cell transfer technology using polymers, the development of biopin to insert peptide-like pins into delivery cells will suggest the possibility as a material for cell delivery and cell therapy and nano-bio applications using them. .

Thus, the present invention provides a membrane protein;

One or more cell-penetrating peptides (CPPs) bound to the protein; And

Provided is a fusion protein comprising one or more cell adhesive peptides bound to the protein.

The fusion protein has a structure in which one or more cell penetrating peptides and one or more cell adhesion peptides are coupled to a portion of the cell membrane protein, and more specifically, the cell penetrating peptides and the cell adhesion peptides may be respectively coupled to both ends of the cell membrane protein. .

As used herein, "biopin" refers to a fusion protein for immobilizing a biological material, for example, a cell. In particular, the three elements used in the biopin consists of a specific membrane protein, a cell penetrating peptide and a cell adhesion peptide of a mammalian cell, the specific membrane protein of the cell is bound to the head, both ends of the membrane protein Cell penetrating peptides and cell adhesion peptides each form a pin structure forming a tip. More specifically, the cell membrane and the cell adhesion peptides are fused to the cell membrane and the intracellular site and the extracellular site. The fusion protein was named biopin. When the biopine is delivered to the cell, the cell penetrating peptide located at the cell membrane and fused to the inner part of the cell is directed toward the inside of the cell, and the cell adhesion peptide fused to the outer part is exposed to the outside of the cell and attached to the target cell or tissue ( 1, see the top drawing). The externally exposed pins are attached to the target cells or tissues to increase intercellular binding. When a fusion protein in the form of a cell-penetrating peptide fused only to the inner domain of the membrane protein cell is delivered to the cell, it is located on the cell membrane but is exposed to the outside of the cell. This is evidenced by the absence of cell adhesion peptides that do not plug into the target cells or tissues (see FIG. 1, bottom figure).

The cell membrane protein is not particularly limited as long as it is a membrane protein of mammalian cells. Preferably, the transmembrane domain is preferable. For example, the cell membrane protein that does not affect cell death, including opioid receptor delta transmembrane domain 1 (OPRD TM1) and the like, is not particularly limited.

The cell-penetrating peptide (CPP) or protein transduction domain (PTD) is a specific protein, virus, Refers to a substance peptide capable of delivering DNA, RNA, fat, carbohydrates or chemical compounds into the cytoplasm or nucleus of eukaryotic or prokaryotic cells.

The cell penetrating peptide is not particularly limited, but Ant- (Antennapedia), a trans-activating transcriptional activator (Tat) protein derived from human immunodeficiency virus type I (HIV-1), an antennapedia homeodomain of Drosophila Or penetratin peptide, Mph-1 of mouse transcription factor, VP22 of HSV-1 and HP4 of herring protamine.

In addition, the cell penetrating peptides may comprise cell-specific PTDs having specificity for a particular cell type or condition. One example of such a cell-specific PTD is the Hn1 synthetic peptide described in US Patent Publication 2002-0102265.

In addition, the cell penetrating peptide may be a synthetic peptide. An example of such a synthetic peptide is a peptide in which Ho et al . Modified 47-57 residues of Tat protein to optimize the protein delivery ability of HIV's Tat protein (Ho et. al . (2001) Cancer Res 61 (2): 474-7).

In a specific embodiment of the invention, the cell penetrating peptide comprises Tat: YGRKKRRQRRR (SEQ ID NO: 2) of HIV.

The cell adhesion peptide is a bioactive compound used covalently to the scaffold to enhance cell adhesion or biospecific cell adhesion when using the scaffold as a mechanical support for cell growth and tissue formation in tissue engineering and wound treatment. By ligand, SEQ ID NO: 3 (RGDS), SEQ ID NO: 4 (KQAGDV), SEQ ID NO: 5 (VAPG), SEQ ID NO: 6 (YAVTGRGDSPAS, FIB1), SEQ ID NO: 7 (ATLQLQEGRLHFXFDLGKGR, EF1zz), SEQ ID NO: 8 (AGTFALRGDNPQG , A99) or amino acid sequence of SEQ ID NO: 9 (GEFYFDLRLKGDKY, 531), or an amino acid sequence comprising at least one or more of the amino acid sequence, for example, amino acid sequence of SEQ ID NO: 10 (GRGDSP) can be used, but is not particularly limited thereto. It is not.

According to one embodiment of the present invention, the fusion protein of the present invention may be in a form in which the cell penetrating peptide and the cell adhesion peptide are bound to both ends of the cell membrane protein having the amino acid sequence of SEQ ID NO: 1.

Fusion proteins of the present invention can be prepared by methods known in the art for synthesizing peptides. For example, it may be prepared by a method of synthesis in vitro through genetic recombination and protein expression systems or peptide synthesizers.

Accordingly, the present invention provides a recombinant vector expressing the fusion protein.

As used herein, a "recombinant vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell and comprises an essential regulatory element operably linked to express a gene insert.

Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. Further, the expression vector includes a selection marker for selecting a host cell containing the vector, and includes a replication origin in the case of a replicable expression vector.

The recombinant vector of the present invention may be prepared by inserting a nucleic acid encoding a cell membrane protein into a general E. coli strain expression vector, pHis / TAT. In a preferred embodiment of the present invention, although pHis / TAT was used as the expression vector for E. coli, the expression vector is not necessarily limited thereto. All E. coli expression vectors that can be generally used may be used without limitation.

In a preferred embodiment of the present invention, the OPRD TM1 domain coding gene (SEQ ID NO: 1), the cell penetrating peptide coding gene (SEQ ID NO: 2) and the cell adhesion peptide coding gene of the present invention using a pHis / TAT vector, which is a vector for E. coli strain expression A recombinant vector was prepared by inserting the DNA fragment containing (SEQ ID NO: 10) (see Fig. 2).

The invention also provides a transformant transduced with a recombinant vector expressing the fusion protein of the invention.

The transformation may include any method of introducing a nucleic acid into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique according to the host cell as known in the art. These methods include electroporation, plasma fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation with silicon carbide fibers, agrobacterial mediated transformation, PEG, dextran sulfate, lipofect Such as but not limited to.

In addition, since the expression amount of the protein and the expression are different depending on the host cell, the host cell most suitable for the purpose may be selected and used.

Examples of host cells include Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus but are not limited to, prokaryotic host cells such as mirabilis or Staphylococcus . In addition, fungi (e.g., Aspergillus ), yeast (e. G., Pichia < pastoris), Mai access to the Yasaka Auxerre Vichy kids (Saccharomyces cerevisiae), investigating car break in Rome Seth (Schizosaccharomyces), Castello La Neuro Chrysler Corporation (Neurospora can be used lower eukaryotic cells, insect cells, plant cells, mammalian cells, such as in higher eukaryotic origin, including such as crassa)) as a host cell.

The transformant can be easily prepared by introducing the recombinant vector into any host cell. In a preferred embodiment of the present invention, a transformant was prepared by introducing the recombinant vector pHis / TAT into E. coli strain BL21 (DE3).

The present invention also provides a method for producing a fusion protein comprising the step of separating and purifying a culture of a transformant transduced with a recombinant vector expressing the fusion protein of the present invention.

The fusion protein is preferably purified by culturing the transformant according to a conventional culture method. The fusion protein may have any modification to a part of the amino acid sequence to the extent that does not affect the cytokine production capacity depending on the insert introduced into the recombinant vector, that is, the coding sequence. By modification is meant modification by deletion, insertion or substitution.

The invention also provides polyclonal antibodies that specifically bind to the fusion proteins of the invention.

Although the manufacturing method of the said polyclonal antibody is not specifically limited, It is preferable to manufacture according to the following method.

The fusion protein of the present invention is immunized by one to several injections into SPF (specific pathogen free) animals. After a certain time after the final immunization, whole blood is taken and serum is obtained to obtain polyclonal antibodies to the proteins of the invention.

The immunized animal is not particularly limited as long as it is an animal normally used for immunization, but is preferably a rat. The number, duration and method of administration for the immunization are not particularly limited because they can be modified or changed at the level of those skilled in the art.

The invention also relates to cell membrane proteins;

One or more cell-penetrating peptides (CPPs) bound to the protein; And

One or more cell adhesive peptides bound to the protein;

The cell penetration peptide relates to a fusion protein to which a fluorescent substance is bound.

The fluorescent material can be linked directly or indirectly using other mediators by chemical, physical covalent or non-covalent association with the cell penetrating peptide.

The fluorescent material is not particularly limited, but Dylight 488 NHE-ester dye, VybrantTM DiI, VybrantTM DiO, quantum dots nanoparticles, fluorosane, rhodamine, lucifer yellow, B-phytoerythrin, 9-a Chridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3- (4'-iso Thiocyatophenyl) -4-methylcoumarin, succinimidyl-pyrenebutyrate, 4-acetamido-4'-isothiocyanatostilben-2,2'-disulfonic acid derivative, LC ™ -Red 640 , LC ™ -Red 705, Cy5, Cy5.5, lysamine, isothiocyanate, erythrosine isothiocyanate, diethylenetriamine pentaacetate, 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-sothitoudinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, 9-isothiocyanatoacrylic , Credin Orange 9-I (Soti (2-benzoxazoylyl) phenyl) melimide saradiazole, stilbene, pyrene, ibendomer, silica containing fluorescent substance, group II / IV semiconductor quantum dots, III / Group V semiconductor quantum dots, group IV semiconductor quantum dots, or an isocomponent hybrid structure, preferably, the fluorescent material may include quantum dot nanoparticles, Cy3.5, Cy5, Cy5.5, Cy7, ICG ( indocyanine green), Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, IRDye82, Cresy Violet, Nile Blue, Oxazine 750, Rhodamine800, lanthanide series and Texas Red, In the case of the quantum dots nanoparticles, group II-VI or group III-V compounding may be used. In this case, the quantum dot nanoparticles may be selected from the group consisting of CdSe, CdSe / ZnS, CdTe / CdS, CdTe / CdTe, ZnSe / ZnS, ZnTe / ZnSe, PbSe, PbS InAs, InP, InGaP, InGaP / ZnS and HgTe More than one can be used.

The fusion protein according to the present invention can provide targeting to the fusion protein by introducing tissue specific binding components either directly or by other mediators by chemical, physical covalent or non-covalent binding to the cell penetrating peptide.

The tissue-specific binding component is not particularly limited, but antigen, antibody, RNA, DNA, hapten, avidin (avidin), streptavidin (neutravidin), protein A, protein G, lectin (lectin), selectin, radioisotope labeling components, tumor markers, etc. may be used alone or in combination of two or more.

Alternatively, the cell penetrating peptide may be cell-specific PTD having specificity for a specific cell type or condition, for example, the fusion protein of the present invention may be targeted, including the Hn1 synthetic peptide described in US Patent Publication No. 2002-0102265. Can have

The fusion protein according to the invention can be used for the treatment of diseases by introducing pharmaceutically active ingredients directly or by other mediators by chemical, physical covalent or non-covalent binding to the cell penetrating peptides.

The pharmaceutically active ingredient is not particularly limited, but siRNA, antisense, anticancer agents, antibiotics, hormones, hormonal antagonists, interleukin, interferon, growth factor, tumor necrosis factor, endotoxin, lymphokoxy, urokinase, streptokinase, tissue plasminogen activator , Protease inhibitors, alkylphosphocholines, radioisotope-labeled components, cardiovascular drugs, gastrointestinal drugs, or nervous system drugs may be used alone or in combination.

The anticancer agent is not particularly limited thereto, for example, epirubicin, docetaxel, gemcitabine, paclitaxel, cisplatin, carboplatin, Taxol, procarbazine, cyclophosphamide, diactinomycin, daunorubicin, etoposide, tamoxifen doxorubicin ), Mitomycin, mitomycin, bleomycin, plecomycin, transplatinum, vinblastin, methotrexate, and the like can be used.

The present invention also relates to a fusion protein according to the present invention to which a fluorescent substance is bound; And

It relates to a contrast agent composition comprising a pharmaceutically acceptable carrier.

The fusion protein of the present invention can be used as a contrast agent capable of imaging the target site through the magnetic resonance and optical imaging device because the fluorescent material is physically coupled.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical field and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide But are not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool.

In addition, the contrast agent composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

In one embodiment, the contrast agent composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline solution Can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Another preferred embodiment of the contrast agent composition of the present invention may be in the form of a sterile injectable preparation of a sterile injectable aqueous or oleaginous suspension. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e. G., Tween 80) and suspending agents.

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (for example, a solution in 1,3-butanediol). Vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. For this purpose, any non-volatile oil including synthetic mono or diglycerides and less irritant may be used.

The contrast agent composition of the present invention may be used to obtain an image by detecting a signal emitted from a fluorescent fusion protein by administering to a tissue or cell isolated from a diagnosis subject.

In order to detect a signal emitted by the fluorescent fusion protein, it is preferable to use a magnetic resonance imaging apparatus (MRI) and optical imaging.

A magnetic resonance imaging apparatus puts a living body in a strong magnetic field and irradiates a radio wave of a specific frequency to absorb energy into an atomic nucleus such as hydrogen in a biological tissue to make the state high in energy, And the energy is converted into a signal, processed by a computer, and imaged. Since the magnetic or radio waves are not disturbed by the bone, a sharp three-dimensional tomographic image can be obtained at the end, transverse, or any angle with respect to the hard bone around or the brain or the bone marrow. In particular, the magnetic resonance imaging apparatus is preferably a T2 spin-spin relaxation magnetic resonance imaging apparatus.

The present invention also relates to a fusion protein according to the present invention to which a fluorescent substance and a tissue specific binding component are bound; And

A directed directed contrast composition comprising a pharmaceutically acceptable carrier.

In the fusion protein of the present invention, fluorescent materials are bound to have fluorescence and tissue-specific binding components are bound to the cell penetrating peptides so that they can be targeted, and imaging of the target site is possible through magnetic resonance and optical imaging devices. It can be used as a contrast medium.

The present invention also provides a fusion protein conjugated with a fluorescent substance and a pharmaceutically active ingredient; And

A contrast diagnostic or therapeutic contrast composition comprising a pharmaceutically acceptable carrier.

The fusion protein of the present invention binds to the pharmaceutically active ingredient physicochemically and simultaneously contains the fluorescence of the fusion protein. Thus, nano probes and drugs or genes such as separation, diagnosis or treatment of biological molecules through magnetic resonance and optical imaging devices, etc. It can be used for a delivery vehicle or the like.

As a representative example of the biological diagnosis using the fusion protein, molecular magnetic resonance imaging or a magnetic relaxation sensor may be mentioned. As the size of the fusion protein increases, it shows a larger T2 contrast effect, which can be used as a sensor for detecting biomolecules. That is, when a specific biomolecule induces fusion protein fusion, T2 magnetic resonance imaging effect is increased. Biomolecules are detected using these differences.

In addition, the fusion proteins of the present invention diagnose and / or treat various diseases associated with tumors, such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer. It can be used to

More specifically, tumor cells express and / or secrete certain substances which are produced little or no in normal cells, which are generally termed "tumor markers". A fusion protein made by binding a substance capable of specifically binding such a tumor marker to a cell penetrating peptide of the fusion protein may be usefully used for tumor diagnosis. There are a variety of tumor markers in the art as well as materials that are capable of specifically binding to them.

In addition, the tumor markers may be classified into ligands, antigens, receptors, and nucleic acids encoding them according to a mechanism of action.

When the tumor marker is "ligand", a substance capable of specifically binding to the ligand can be introduced into the fusion protein according to the present invention, but a receptor capable of specifically binding to the ligand Or the antibody will be suitable. Examples of ligands and receptors that can specifically bind to the present invention include synaptotagmin C2 (synaptotagmin C2) and phosphatidylserine, annexin V and phosphatidylserine, integrin and its Receptors, Vascular Endothelial Growth Factor (VEGF) and its receptors, angiopoietin and Tie2 receptors, somatostatin and its receptors, vasointestinal peptides and their receptors. It is not.

A representative example where the tumor marker is a "receptor" is a folic acid receptor expressed in ovarian cancer cells. Substances that can specifically bind to the receptor (folic acid in the case of folic acid receptors) can be introduced into the fusion protein according to the invention, and ligands or antibodies that can specifically bind to the receptor will be suitable.

When the tumor marker is an "antigen", a substance capable of specifically binding to the antigen can be introduced into the fusion protein according to the present invention, and an antibody capable of specifically binding to the antigen will be suitable. Examples of antigens and antibodies that specifically bind to the present invention include carcinoembryonic antigens (colon cancer marker antigens), Herceptin (Genentech, USA), and HER2 / neu antigens (HER2 / neu antigens-breast cancer markers). Antigen) and Herceptin, prostate-specific membrane antigen (prostate cancer marker antigen) and rituxan (IDCE / Genentech, USA).

Such antibodies can be obtained commercially or prepared according to methods known in the art. Generally mammals (eg, mice, rats, goats, rabbits, horses or sheep) are immunized one or more times with an appropriate amount of antigen. After a period of time when the titer reaches an appropriate level, it is recovered from the serum of the mammal. The recovered antibody can be purified using known processes if desired and stored in a frozen buffered solution until use. Details of this method are well known in the art.

On the other hand, "nucleic acid" includes RNA and DNA encoding the aforementioned ligand, antigen, receptor or part thereof. Nucleic acid having a specific base sequence can be detected using a nucleic acid having a base sequence complementary to the base sequence because the nucleic acid has a feature that forms a base pair between complementary sequences as known in the art . A nucleic acid having a nucleotide sequence complementary to the nucleic acid encoding the enzyme, ligand, antigen, receptor can be introduced into the fusion protein according to the present invention.

In addition, the nucleic acid has a functional group such as -NH ₂ , -SH, -COOH at the 5'- and 3'- terminal can be usefully used to bind to the cell penetrating peptide.

Such nucleic acids can be synthesized by standard methods known in the art, for example using automated DNA synthesizers (such as those available from BioSearch, Applied Biosystems, etc.). By way of example, phosphorothioate oligonucleotides are described in Stein et. al . Nucl . Acids Res . 1988, vol. 16, p. 3209). Methylphosphonate oligonucleotides can be prepared using controlled free polymer supports (Sarin et. al . Proc . Natl . Acad . Sci . USA 1988, vol. 85, p.7448).

The present invention also relates to a fusion protein according to the present invention to which a fluorescent substance is bound; And

A multiple diagnostic probe comprising a diagnostic probe is provided.

The diagnostic probe may be a T1 magnetic resonance imaging probe, an optical diagnostic probe, a CT diagnostic probe, or a radioactive isotope.

For example, when the T1 magnetic resonance imaging diagnostic probe is coupled to a fluorescent fusion peptide, the multiple diagnostic probe may simultaneously perform T2 magnetic resonance imaging and T1 magnetic resonance imaging, and when the optical diagnostic probe is combined, magnetic resonance imaging and optical imaging may be performed. At the same time, CT diagnostic probes can be combined to perform MRI and CT diagnosis at the same time. In addition, when combined with radioisotope, MRI, PET, and SPECT can be performed simultaneously.

The T1 magnetic resonance imaging diagnostic probe may include a Gd compound or a Mn compound, and the optical diagnostic probe may be an organic fluorescent dye (dye), a quantum dot, or a dye labeled inorganic support (eg SiO ₂ , Al _2). O ₃ )), CT diagnostic probes include I (iodine) compounds, or gold nanoparticles, and radioisotopes include In, Tc, F, and the like.

The invention also relates to fusion proteins according to the invention; And

It relates to a cell therapy comprising at least one stem cell or immune cell.

When the fusion protein of the present invention is combined with stem cells or immune cells by at least one cell penetrating peptide and delivered into a cell, the fusion protein is fixed to a cell membrane of a target cell, tissue, or organ to fix the stem cells or immune cells for a long time. It is effective to increase the efficiency of cell therapy.

In addition, the fusion protein of the present invention can be cell traced by measuring the fluorescence intensity when the fluorescent material is bound and delivered into the cell by direct or other media by chemical, physical covalent or non-covalent bond.

In addition, the fusion protein of the present invention can enhance cell targeting when the tissue-specific binding component is delivered to the cell by direct or other media by chemical, physical covalent or non-covalent binding.

In addition, the fusion protein of the present invention, by chemical or physical covalent or non-covalent binding, by using a direct or other mediator, a pharmaceutical active ingredient, for example, a drug for inhibiting or treating a cardiovascular disease, a brain disease, or the like is combined into a cell. When delivered, the therapeutic effect of the cells can be enhanced.

The invention also relates to fusion proteins according to the invention; And

It relates to a medical stent comprising at least one endothelial cell or vascular progenitor cell.

When the fusion protein of the present invention is combined with endothelial cells or vascular progenitor cells by at least one cell penetrating peptide and delivered into cells, the fusion protein is fixed to the cell membranes of target cells, tissues, or organs so that the endothelial cells or vascular progenitor cells, etc. By fixing, there is an effect of increasing the treatment efficiency of the medical stent.

In addition, the fusion protein of the present invention can be stent tracking by measuring the fluorescence intensity when the fluorescent material is bound and delivered into the blood vessels by direct or other media by chemical, physical covalent or non-covalent bonds.

In addition, the fusion protein of the present invention can increase the target orientation when the tissue-specific binding component is delivered to the blood vessel by direct or other media by chemical, physical covalent or non-covalent binding.

In addition, the fusion proteins of the present invention can be used for the therapeutic treatment of stents when pharmacologically active ingredients, such as cytostatic drugs, are delivered to the blood vessels by means of chemical, physical covalent or non-covalent bonds, either directly or using other mediators. The effect can be enhanced.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the scope of the present invention is not limited by the following Examples.

Example 1 Preparation of Fusion Protein (Biopin) According to Recombination Method

pHis / TAT expression vector (Kwon JH et al ., 2007, Biochem Biophys Res Commun ., 363, 399-404). The OPRD by the cDNA as a template using two primers, Primer-1, 5'-TCGTCC CATATG GGCCGCGGCGACAGCCCGGGATCCCTGGCAATCGCCATCACC-3 '( Nde I site underlined) and Primer-2, 5'-AGGCATCTCGAGTTAGCGGCGGCGCTGGCGGCGTTTCTTGCGGCCGTAAGTGTACCGGACGATGCCGAA- 3' ( Xho I site underlined) PCR fragments were obtained, DNA fragments were digested with restriction enzymes ( Bam HI / Xho I), put into pHis / TAT expression vectors, expressed in Escherichia coli BL21 (DE3), and the proteins were separated and purified using Ni-NTA affinity chromatography. . In this case, the peptide is composed of peptides consisting of 31 amino acids from the 48th amino acid (leucine, L), the first domain part of the OPRD protein consisting of 7 transmembrane domains of 372 amino acids, to the 78th amino acid (threonine, T). Bioffin (BPin-RGD) was prepared in which the adhesive peptide (GRGDSP) and the TAT cell penetrating peptide were fused at the terminal and C-terminus, respectively (see FIG. 2). At this time, in order to prepare the cell infiltration peptide on only one side of the OPRD peptide composed of 31 amino acids as a control, Primer-1 was prepared by PCR in place of 5'-TCGTCC CATATG GGATCCCTGGCAATCGCCATCACC-3 '(underlined Nde I site).

Example 2 Preparation of Fusion Protein (Biopin) Through Peptide Synthesis

Fusion proteins of the present invention can be prepared using a peptide synthesizer. FIG. 3 is a biosynthetic peptide synthesized by Bio-Synthesis (USA), and a bioffine (BPin-RGD) and a cell penetrating peptide are fused to one side of the OPRD first domain by fusion of a cell adhesion peptide and a cell penetrating peptide. The sequence for preparing BPin-44 is shown.

Example 3 Delivery of Biopin Modified with Fluorescent Cells

Rat cardiac myoblast H9c2 cells incubated in DMEM medium were placed in microtubes and treated with CellMask ™ Plasma Membrane Stains (Invitrogen, USA) at a final concentration of 5 μg / mL and left in a 37 ° C. CO ₂ incubator for 30 minutes. It was. Thereafter, centrifugation and washing with phosphate-buffered saline (PBS) three times, and resuspended in 500 μl of DMEM medium were prepared.

Modifications of the two peptides (BPin-44, BPin-RGD) were performed as follows. DyLight405 NHS Ester (Thermo, USA) was dissolved in 100 μl of dimethylformonamide and 0.1 mg of each peptide was added thereto. After reacting at room temperature for 1 hour, dialysis (Slide-A-Lyzer Mini Dialysis Units, Thermo, USA) was performed to remove the unmodified dye.

Each peptide modified in cells prepared by staining (5 × 10 ⁴ cells) was treated with 100 nM and then left in a 37 ° C. CO ₂ incubator for 1 hour. After centrifugation and PBS washing was repeated three times.

For observation using a confocal microscope, the cell chamber was treated with a mounting solution (3 drops) followed by CellMask ™ Plasma Membrane Stains (red) using an LSM700 confocal microscope (Carl Zeiss, Germany) (Abs 554). / Em 567 nm), Dylight 405 NHE-ester (blue) (Abs 400nm / Em420nm) wavelength was observed.

As shown in Figure 4, it was confirmed that both peptides are located in the cell membrane.

Example 4 Increasing Intercellular Binding by Biopin

Rat cardiac myoblasts (Rat cardiac myoblast H9c2 cells) were incubated 100% in a 4-well chamber (Lab-Tek chamber slide). CellMask ™ Plasma Membrane Stains (Invitrogen, USA) was prepared by treatment at a concentration of 5 μg / mL for 30 minutes in a 37 ° C. CO ₂ incubator and then washed three times with PBS.

Rat mesenchymal stem cells (MSCs) were cultured in a Petri dish with MesenPRO (Gibco, USA) medium and then trypsin-EDTA and trypsin-neutralizing solution in a 1: 1 ratio. The cells were separated for 5 minutes, treated with a cell-modified dye (VybrantTM DiO, Invitrogen, USA) at a final concentration of 2 μl / mL, reacted for 30 minutes in a 37 ° C CO ₂ incubator, washed three times with PBS, and washed at 3,200 rpm. Prepared by centrifugation for a minute.

Here, the bioffin prepared in Example 1 was treated with a final 100 nM concentration and then reacted by shaking left and right for 1 hour in a 37 ° C. CO ₂ incubator, washed three times with PBS, and centrifuged to contain 10% FBS. Resuspended by treating 400 μL of DMEM medium. It was sprinkled onto the stained H9c2 cells and left for 10 minutes in a 37 ℃ CO ₂ incubator. After washing several times with PBS to remove MSCs not attached to H9c2, 200 μl of 0.1% Triton X-100 was added to quantify the attached MSCs, and after 5 minutes, removed by fluorescence spectrometer Victor3 (Fluorescent Spectrometer Victor3, Perkin- Elmer, USA) was used to measure the wavelength at Abs 484 nm / Em 501 nm.

As shown in FIG. 5, it was confirmed that the intercellular binding was significantly increased in the experimental group to which the biopin BPin-RGD was delivered.

Attach an electronic file to a sequence list

Claims (20)

Cell membrane proteins;
One or more cell-penetrating peptides (CPPs) bound to the protein; And
A fusion protein comprising one or more cell adhesive peptides bound to the protein.
The method of claim 1,
The cell membrane protein is a fusion protein comprising opioid receptor delta transmembrane domain 1 (OPRD TM1) of the opioid receptor delta.
The method of claim 1,
The cell penetrating peptide is at least one fusion protein selected from the group consisting of trans-activating transcriptional activator (Tat), Antp, Mph-1, VP22 and HP4.
The method of claim 1,
Cell adhesion peptides include SEQ ID NO: 3 (RGDS), SEQ ID NO: 4 (KQAGDV), SEQ ID NO: 5 (VAPG), SEQ ID NO: 6 (YAVTGRGDSPAS, FIB1), SEQ ID NO: 7 (ATLQLQEGRLHFXFDLGKGR, EF1zz), SEQ ID NO: 8 (AGTFALRGDNPQG, A99 Or the amino acid sequence of SEQ ID NO: 9 (GEFYFDLRLKGDKY, 531), or an amino acid sequence comprising at least one amino acid sequence.
The method of claim 1,
The fusion protein is a fusion protein in which a cell penetrating peptide and a cell adhesion peptide are bonded to both ends of a cell membrane protein having the amino acid sequence of SEQ ID NO: 1.
A recombinant vector expressing the fusion protein according to claim 1.
A transformant transduced with the recombinant vector according to claim 6.
A method for preparing the fusion protein of claim 1, comprising separating and purifying the fusion protein from the culture of the transformant according to claim 7.
Cell membrane proteins;
One or more cell-penetrating peptides (CPPs) bound to the protein; And
One or more cell adhesive peptides bound to the protein;
A fusion protein in which the fluorescent substance is bound to the cell penetrating peptide.
10. The method of claim 9,
Fluorescent materials include Dylight 488 NHE-ester dye, VybrantTM DiI, VybrantTM DiO, quantum dots nanoparticles, Cy3.5, Cy5, Cy5.5, Cy7, indocyanine green, Cypate, ITCC, NIR820, NIR2, At least one fusion protein selected from the group consisting of IRDye78, IRDye80, IRDye82, Cresy Violet, Nile Blue, Oxazine 750, Rhodamine 800, Lanthanide and Texas Red.
10. The method of claim 9,
A fusion protein to which the cell-penetrating peptide is further bound to tissue-specific binding components.
12. The method of claim 11,
Tissue-specific binding components include antigen, antibody, RNA, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin, selectin ( at least one selected from the group consisting of selectin), a radioisotope label, and a substance capable of specifically binding to a tumor marker.
10. The method of claim 9,
A fusion protein to which the cell penetrating peptide further comprises a pharmaceutically active ingredient.
The method of claim 13,
Pharmaceutically active ingredients include siRNA, antisense, anticancer agents, antibiotics, hormones, hormonal antagonists, interleukins, interferons, growth factors, tumor necrosis factors, endotoxins, lymphotoxins, urokinase, streptokinase, tissue plasminogen activators, protease inhibitors, alkylposses A fusion protein that is one or more selected from the group consisting of pocholine, a radioisotope labeled component, a cardiovascular drug, a gastrointestinal drug, and a nervous system drug.
A fusion protein according to claim 9; And
A contrast agent composition comprising a pharmaceutically acceptable carrier.
A fusion protein according to claim 11; And
A composition comprising a pharmaceutically acceptable carrier.
A fusion protein according to claim 13; And
A contrast agent composition for simultaneous diagnosis or treatment comprising a pharmaceutically acceptable carrier.
Comprising a fusion protein and a diagnostic probe according to claim 9,
The diagnostic probe is a multiple diagnostic probe that is a label for reading the image bound to the cell penetrating peptide of the fusion protein.
A fusion protein according to claim 1; And at least one stem cell or immune cell,
The fusion protein is a cell therapy agent bound to the cell membrane of stem cells or immune cells.
A fusion protein according to claim 1; And at least one endothelial cell or vascular progenitor cell, wherein the fusion protein is bound to the cell membrane of the endothelial cell or vascular progenitor cell and is fixed to the stent.

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