KR101629362B1 - Composition for Cancer Diagnosis and Therapy Using Leucine Zipper Pair - Google Patents

Abstract

The present invention relates to a composition for diagnosing and treating cancer using a leucine zipper pair (ZR / ZE), more specifically, a cancer diagnostic fusion protein in which an antibody specific for a cancer cell surface receptor and a ZR peptide are fused; And a composition for diagnosis and treatment of cancer which can selectively treat cancer after cancer diagnosis, which comprises a nanoemulsion for treating cancer, wherein the ZE peptide is immobilized on the surface and contains genes or medicines for cancer treatment therein will be.
The composition for diagnosing and treating cancer of the present invention can detect only specific cancer cells using a cancer diagnostic fusion protein in which a ZR peptide is fused with an antibody specific for a cancer cell surface receptor and the fusion protein and the ZE peptide are immobilized on the surface ZR / ZR leucine zipper pairing of cancer therapeutic nanoemulsion has the advantage of transferring therapeutic substance only to specific cancer cells. In addition, since the fusion protein or nanoemulsion can be immobilized with a fluorescent substance, it is possible to perform multimodal analysis, and thus it is possible to provide a fusion protein or a nano-emulsion in which cancer cells And can be usefully used for the development of effective diagnosis and therapeutic agents.

Description

Technical Field [0001] The present invention relates to a composition for diagnosing and treating cancer using a leucine zipper pair,

The present invention relates to a composition for diagnosing and treating cancer using a leucine zipper pair (ZR / ZE), more specifically, a cancer diagnostic fusion protein in which an antibody specific for a cancer cell surface receptor and a ZR peptide are fused; And a composition for diagnosis and treatment of cancer which can selectively treat cancer after cancer diagnosis, which comprises a nanoemulsion for treating cancer, wherein the ZE peptide is immobilized on the surface and contains genes or medicines for cancer treatment therein will be.

It is known that an antibody or antibody fragment can be conjugated to a radioactive isotope, drug or toxin to apply a diagnostic or therapeutic principle to a tumor or lesion site. The biggest obstacle to using this method was that it was difficult to load a sufficient amount of therapeutic or diagnostic agent into the antibody. Another problem is that when the antibody is overloaded with a therapeutic or diagnostic agent, the human body may reject and destroy the antibody conjugate.

It is known in the art to conjugate cytotoxic drugs to antibodies to achieve therapeutic results by targeting, for example, it is known that methotrexate (MTX) can be conjugated to antibodies and some selective cytotoxicity is observed . It is desirable to enhance the cytotoxicity of such conjugates by increasing the loading of cytotoxic agents. However, multiple conjugation of an individual drug molecule to an antibody ultimately reduces its immunoreactivity, which is observed when more than about 10 molecules are loaded.

Also, techniques have been studied in which a drug is conjugated to a polymer carrier and then conjugated to an antibody, and this technique has the advantage that a larger number of drug molecules can be delivered to a target site. Ryser et al. (Ryser et al., Proc. Nat.l Acad. Sci., 75: 3867, 1978) reported that only about 3 MTX could be loaded per carrier using polylysine as a polymer carrier, Is lowered. The high content of amines in the polymer, which are in the form of ammonium groups predominantly charged, adversely affects the selectivity of the cytotoxic effect by causing the conjugate to adhere to normal cells.

Recently, theragnostic agents for diagnosis and treatment have been developed. These are usually made of small-sized materials, and usually have a form in which fluorescent dyes and radioactive molecules are carried on the inside of liposomes, polymers and nanoparticles, and drugs or diagnostic markers are introduced to the outside. Recently, the synthesis of therapeutic contrast material composed of lipid structure with excellent biocompatibility has been mainly studied. Especially, microbubble ultrasound agent (microbubble), which is a micrometer-sized air- , And MB) have been actively studied (David C., Eur J Radio l., 60: 324, 2006). These studies have recently focused primarily on the application of properties such as non-toxic, high-water-soluble, or easy surface modification for applications in the biotechnology field of nanomaterials, which are now referred to as "nano-bio fields" It is set.

The microbubbles based on the known lipid structures can be used for various biocompatibility in vivo or in vitro studies as well as diagnosis by imaging analysis of blood vessels or cancer tissues (Katherine F. et al. , Annual Review of Biomedical Engineering , 9: 415, 2007). However, ultrasound contrast analysis through microbubbles is insufficient for accurate diagnosis of cancer due to low resolution and nonspecific properties. Therefore, by using ultrasonic imaging as well as ultrasound imaging, At the same time, studies have been conducted to develop multimodal imaging materials through fluorescence and ultrasound, which allow detailed distribution through fluorescence image analysis (Bart G. et al., Journal of Controlled Release, 152: 249, 2011 ).

However, most of the known substances still remain in the target nonspecific contrasting effect, so that they can be used only for the linear diagnosis of tissues such as blood vessels and lymphatic vessels, or the therapeutic gene or drug is delivered only to lesion cells (Wang C. et al., Biomaterials , 33: 1939, 2012). In order to support the therapeutic substance, it is necessary to form a hybrid with another structure.

On the other hand, leucine zipper is a kind of high-order structure common to a specific DNA binding protein. It is a protein C / EBP that binds to a CAAT box of a nucleotide sequence on a gene transcribed by RNA polymerase II, It is known as a protein characterized by a protein CREB that binds to a specific region of DNA, a transcription factor of yeast, GCN4, a nuclear localization of cancer, Myc, Fos, and Jun proteins (Vinson et al., Science , 246 : 911 1989; Dmitry Krylov and Charles R Vinson, Encyclopedia of Life Sciences, 1, 2001). Near the C-terminus of these proteins, the leucine residue is repeated every seven amino acids. This region forms the α-helical structure, so that the leucine is arranged on one side every two revolutions of the α-helical structure. Therefore, when two such proteins are present, the leucine residues arranged on the side are combined with each other like a zipper by a small number of bonds to form a bimodule. A basic amino acid-rich region exists at the N-terminal side of the basic structure of the leucine zipper, and this region becomes a base-region leucine zipper (B-Zip) by the formation of a biosynthetic entity.

Among them, the ZE / ZR heterodimeric leucine zipper pair is derived from the B-Zip homodimer of the vitellogenin-binding protein (VBP), a precursor of egg yolk protein present in the female blood , Acid residues ZE (acidic peptide ZE) and basicity (ZE) were changed to glutamic acid or arginine, respectively, by replacing all the residues located at e and g in the first four portions of the seven portions of the ZE / ZR leucine zipper pair with glutamic acid or arginine, Peptide peptide ZR (Moll, JR et al. , Protein Sci., 10: 649, 2001).

Accordingly, the present inventors have made efforts to develop a cancer diagnosis and cancer cell-specific therapeutic system by using the ZE / ZR leucine zipper pair to improve the problems of the conventional drug delivery system for treating cancer, When a composition for diagnosing and treating cancer comprising a specific antibody, a ZR peptide fusion protein and a ZE peptide-immobilized nanoemulsion binding to the ZR peptide is used, the therapeutic substance can be effectively delivered to only specific cancer cells by targeting cancer cells And the present invention was completed.

It is an object of the present invention to provide a composition for diagnosing and treating cancer which can specifically diagnose and diagnose cancer cells using ZR / ZE leucine zipper pair binding.

In order to achieve the above object, the present invention provides a cancer diagnostic fusion protein in which a ZR peptide is fused with an antibody specific to a cancer cell surface receptor; And a composition for diagnosing and treating cancer which can selectively treat cancer after cancer diagnosis, which comprises a nanoemulsion for cancer treatment, wherein the ZE peptide is immobilized on the surface and contains a gene or drug for treating cancer, do.

The composition for diagnosing and treating cancer of the present invention can detect only specific cancer cells using a cancer diagnostic fusion protein in which a ZR peptide is fused with an antibody specific for a cancer cell surface receptor and the fusion protein and the ZE peptide are immobilized on the surface ZR / ZR leucine zipper pairing of cancer therapeutic nanoemulsion has the advantage of transferring therapeutic substance only to specific cancer cells. In addition, since the fusion protein or nanoemulsion can be immobilized with a fluorescent substance, it is possible to perform multimodal analysis, and thus it is possible to provide a fusion protein or a nano-emulsion in which cancer cells And can be usefully used for the development of effective diagnosis and therapeutic agents.

Brief Description of the Drawings Fig. 1 is a graph showing the results of analysis of a cancer-diagnosing fusion protein in which cancer-specific surface-receptor-specific antibodies and ZR peptides are fused and ZE peptides are immobilized on a surface, Fig.
2 is a schematic diagram of ZR / ZE leucine zipper pair binding.
FIG. 3 (A) shows SDS-PAGE results showing the expression amount of ZR-Herceptin Fab protein, which is a fusion protein of cancer-specific surface receptor-specific antibody and ZR peptide fusion (1 lane: marker 2 lane : 4D5Fab antibody-ZR fusion protein), and B shows Western blotting results in which the expression amount of ZR-Herceptin Fab protein, which is a fusion protein for cancer diagnosis, in which an antibody specific for cancer cell surface receptor and ZR peptide are fused (lanes 1 - 3: fractions containing the 4D5 Fab antibody-ZR fusion protein 1 to 3).
Fig. 4 is data (C) obtained by quantifying the characteristics of ZE-Rhodamin-PFCE (A), cytotoxicity (B) and ZE-Rhodamin-PFCE of ZE-Rhodamin-PFCE which is a cancer therapeutic nanoemulsion immobilized on the surface of ZE peptide.
FIG. 5 shows data obtained by confirming the binding of the antibody-cell and the ZR / ZE leucine zipper pair with a flow cytometer (FACS).
FIG. 6 is data obtained by confocal microscopy of antibody-cell and ZR / ZE leucine zipper pair binding.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a cancer diagnostic fusion protein in which an antibody specific for a cancer cell surface receptor and a ZR peptide are fused; And a composition for diagnosis and treatment of cancer which can selectively treat cancer after cancer diagnosis, which comprises a nanoemulsion for treating cancer, wherein the ZE peptide is immobilized on the surface and contains genes or medicines for cancer treatment therein will be.

As shown in FIG. 1, the composition for diagnosing and treating cancer of the present invention is characterized in that a cancer-specific fusion protein in which a specific antibody against a cancer cell surface receptor and a ZR peptide are fused specifically binds to a receptor (antigen) Thus, a cancer therapeutic nanoemulsion containing a gene or a drug for cancer treatment, wherein the ZE peptide is immobilized on the surface thereof, is capable of detecting a target cell, and the ZR peptide of the target cell-fusion protein complex formed and the ZE / ZR Leucine zipper pair binding can selectively deliver therapeutic agents to target cells only.

In the present invention, the ZR peptide is represented by the amino acid sequence of SEQ ID NO: 2 (LEI RAAFLRR RNTALRT RVAELRQ RVQRLRN IVSQYET RYGPL) and the ZE peptide is represented by the amino acid sequence of SEQ ID NO: 3 (LEI EAAFLEQ ENTALET EVARLEQ EVQRLEN IVSQYET RYGPL) .

In the present invention, the nanoemulsion is characterized by containing a therapeutic agent containing a gene or drug for cancer treatment.

In the present invention, the cancer is preferably selected from the group consisting of breast cancer, liver cancer, pancreatic cancer, brain cancer, stomach cancer, lung cancer, esophageal cancer, colon cancer, prostate cancer, kidney cancer and ovarian cancer. Respectively. When the breast cancer is targeted, the cancer cell surface receptor is HER2 (Human Epidermal Growth Factor Receptor 2 protein). In the present invention, a cancer diagnostic fusion protein fused with an antibody specific for HER2, which is a surface receptor of breast cancer cells and represented by the amino acid sequence of SEQ ID NO: 1, and a ZR peptide was prepared and used.

In the present invention, breast cancer is targeted arbitrarily in order to confirm the effect on the cancer diagnosis and treatment system using the leucine zipper pair. It is not limited to the diagnosis of breast cancer, and various antibodies specific for cancer cell surface receptor and ZR It is obvious to those skilled in the art that fusion proteins with fused peptides can be used.

In the present invention, the cancer therapeutic nanoemulsion in which the cancer diagnostic fusion protein and the ZE peptide are immobilized on the surface is further combined with a fluorescent substance, and the fluorescent substance is fluorescein, fluorescein chlorotriazinyl fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine dyes and thiadicarbocyanine dyes. However, the present invention is not limited thereto, and fluorescent materials known in the art can be used without limitation. In the present invention, preferably, Cy5, a fluorescein isothiocyanate (FITC) and a rhodamine Red was used.

The term " ZE / ZR Leucine zipper pair "in the present invention means that the ZE peptide and the ZR peptide form a heterodimer (Fig. 2) and the ZE / ZR heterodimeric leucine pair zipper pair) was derived from the B-Zip homodimer of the vitellogenin-binding protein (VBP), a precursor of yolk protein in the blood of the female.

The acidic peptide ZE and the acidic peptide ZE were obtained by changing all the residues located at e and g in the first four portions of the seven portions of the ZE / ZR leucine zipper pair of FIG. 2 to glutamic acid or arginine, It is known to produce basic peptide ZR (basic peptide ZR). Conventional ZE and ZR homodimers are known to have four pairs of electrostatic repulsions. However, due to the above residue change, the ZE / ZR heterodimer has four pairs of attractive salt bridges, . Moll et al. Report that the ZE / ZR leucine zipper system has a heterozygote affinity of 10-15 M similar to the streptavidin / biotin system while the affinity of the heterozygosity is in the micromolar range (Moll, JR et al. Protein Sci., 10: 649, 2001).

HER2 of the present invention is an epidermal growth factor receptor 2 (erbB-2, HER2, Neu, CD340, p185) with a 185 kDa type 1 transmembrane receptor tyrosine kinase ), Which is known to promote differentiation and proliferation of mammalian cells. On the other hand, when the HER2 / neu is abnormally activated in the adult, it is known to be involved in the development of cancer. In particular, 20-30% of patients with breast or ovarian cancer are overexpressing HER2 / neu, which is associated with a poor prognosis of breast, ovarian, or stomach cancer. Thus, Herceptin (or Trastuzumab), an antibody specific for HER2, has been developed for the treatment of breast cancer, which is a highly effective treatment for both early-stage and metastatic HER2-positive breast cancer, It is used as a treatment.

In the embodiment of the present invention, in order to diagnose and treat breast cancer using a composition for diagnosing and treating cancer, fusion of 4D5 antibody and ZR peptide (LEI RAAFLRR RNTALRT RVAELRQ RVQRLRN IVSQYET RYGPL), which is a Fab fragment form as a Herceptin antibody, (Fig. 3). To confirm the binding of cell surface receptors to target breast cancer cell lines and to detect cancer cells later, Cy5 or Fluorescein isothiocyanate (FITC) was added .

In the present invention, a cancer cell surface-specific antibody and a ZR peptide were fused with each other for targeting cancer cells. However, the target substance such as a molecule, a ligand, or a receptor on the surface of a target cell was selectively recognized (DNA or RNA), a protein, an antibody, an antigen, an aptamer (RNA, DNA and a peptide aptamer), a receptor, (S), hormone, streptavidin, avidin, biotin, lectin, ligand, agonist, antagonist, enzyme, coenzyme, inorganic ion, enzyme cofactor, sugar, lipid, enzyme substrate, hapten, neutravidin, (For example, Pt, Pd, Au, Ag, Nb, Ir, Rh and Ru), preferably biotin, streptavidin, avidin, antibody, TAMER, POLYPEPTIDE, PEPTIDE, Ligands, receptors, lectins, sugars, lipids, glycolipids or nucleic acids, but are not limited thereto. The target substance refers to a substance in a sample to be separated (or separated and detected or separated, detected and quantified), and specifically includes nucleic acid molecules (DNA or RNA), proteins, peptides, antigens, sugars, lipids, , Viruses, cells, organic compounds, inorganic compounds, metals and inorganic ions. In addition, the sample includes a biological sample, a chemical sample, and an environmental sample, and the biological sample is, for example, blood, plasma, serum, virus, bacteria, tissue, cell, lymph, bone marrow, saliva, milk, urine, But are not limited to, liquid, semen, brain extract, spinal fluid, joint fluid, thymus fluid, ascites fluid, amniotic fluid, cell tissue fluid and cell culture fluid.

In the present invention, in order to confirm the degree of binding with the fusion protein for cancer diagnosis, ZE peptide was bound to rhodamine, which is a basic dye, in the process of preparing a cancer therapeutic nanoemulsion immobilized on the surface, Nanoemulsions can be used not only for treatment but also for multiple image analysis.

A method for producing a cancer therapeutic nanoemulsion in which a ZE peptide is immobilized on a surface thereof comprises the steps of: (a) preparing phosphatidylcholine, cholesterol, DSPE-mPEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- 2-dioleoyl- sn -glycero-3-phosphoethanolamine- N - (lissamine rhodamine B sulphonyl), DSPE-PEG3400- NH ₂ (N- (aminopropylpolyethyleneglycol) carbamyl-distearoylphosphatidyl ethanolamine) (B) dissolving the lipid membrane in distilled water and dispersing it in an ultrasonic wave to prepare a liposome suspension; (c) adding an emulsifier liquid solution And (d) covalently bonding a ZE peptide to the surface of the nanoemulsion prepared as described above. .

In an embodiment of the present invention, a mixture of 73 mol% phosphatidylcholine, 20 mol% cholesterol, 3 mol% DSPE-mPEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- % DPPE-rhodamine (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N - (lissamine rhodamine B sulphonyl) and 2.6 mol% DSPE-PEG3400- NH ₂ (N- (aminopropylpolyethyleneglycol) carbamyl-distearoylphosphatidyl ethanolamine) To prepare a nano emulsion. To bind the ZE peptide to the nanoemulsion prepared in step (d), 10 mM EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) ZE-Rhodamin-PFCE was prepared by covalent coupling with the prepared nano-emulsion.

The nanoemulsion prepared in the above may be used without limitation in the nanoemulsion or nanoliposome known in the art, and any of the methods known in the art may be used. It can be adjusted to a size of 100 nm to 10 탆 according to the kind and concentration of the material to be used, and the size can be controlled also by the cancer treatment agent added according to purpose.

The nanoemulsion may include a film forming material, a phospholipid, a negative charge compound, a compound having an amine group, and a compound having a disulfide group. Examples of the film forming material include 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) Glycero-3-phosphocholine, DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2- dilinoleoyl- , DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn- glycero-3-phosphocholine), DOPC (1,2-dioleoyl- ) Or DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), and phospholipids may be cholesterol or the like. Examples of negative charge carriers include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn- glycero-3-phosphate), DMPA -dimyristoyl-sn-glycero-3-phosphate) or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate). Examples of the compound having an amine group include DPPE glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl- phosphoethanolamine or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); And DSPE-PEG-SPDP {1,2-distearoyl-snglycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) -2000]] may be used as the compound having a disulfide group.

In order to characterize the nanoemulsion prepared in the present invention, the emission peak, the diameter and the polydispersity (the property that the particle size of the dispersed system is not uniform) were determined. As a result, the ZE peptide The emission wavelength peak of the fixed nanoemulsion was 592 nm, the diameter was 123.1 ± 26.9 nm, the polydispersity was 0.087, and it was confirmed that the ZE peptide had a value similar to that of the non-immobilized nanoemulsion. In addition, the cell survival rate was 100% when the nanoemulsion was treated to the breast cancer cell lines SKBR3 and MDF7 and the general breast cell MCF10A by concentration, and it was confirmed that there was no toxicity (Fig. 4).

ZE-Rhodamin-PFCE was added to the target cell-4D5 Fab-ZR fusion protein complex to confirm that the 4D5 Fab antibody-ZR fusion protein prepared in the present invention was targeted to HER2 overexpressed cells (SKBR3) The binding (ZR-ZE) was confirmed by flow cytometry (FACS) experiments. The control group of the fusion protein used a fusion protein in which Fab fragments other than the HER2 specific antibody were fused with the ZE peptide, and MCF7, which is a breast cancer cell line in which HER2 was hardly expressed, was used as a control group of cells. As a result, the 4D5Fab-ZR fusion protein selectively binds to the SKBR3 cell line overexpressing (FITC labeling) Her2, and ZE-Rhodamin-PFCE forms a zipper pair with the ZR peptide to bind to the target cell-4D5Fab-ZR fusion protein complex (Fig. 5).

In addition, it was confirmed that 4D5Fab-ZR fusion protein binds only to SKBR3 cells by using MCF10A and SKBR3 cells, which are general breast cells, in the same manner as the above experiment. ZE-Rhodamin- Peptide and zipper pairs were formed and confirmed to bind to the target cell-4D5Fab-ZR fusion protein complex (Fig. 6).

That is, it was confirmed that the composition for diagnosis and treatment of cancer using the leucine zipper pair of the present invention can specifically detect only the cancer cells to be targeted, and the ZE peptide was fixed on the surface through ZR / ZE leucine zipper pair binding It was confirmed that therapeutic substance can be delivered only to specific cancer cells by nanoemulsion (including therapeutic agent) for cancer treatment.

Accordingly, using the composition for diagnosing and treating cancer of the present invention, there is provided a method for detecting cancer, comprising the steps of: (a) detecting cancer cells using a cancer diagnostic fusion protein in which an antibody specific for a cancer cell surface receptor and a ZR peptide are fused; And (b) treating the cancer by adding a cancer therapeutic nanoemulsion immobilized on a surface of the ZE peptide and transferring the therapeutic agent specifically detected in ZR / ZR leucine zipper pairing with the fusion protein to the cancer cell; And the like.

The composition for diagnosing and treating cancer of the present invention can be useful for targeting and diagnosis of cancer cells or cancer tissues and can be formulated into an oral or parenteral administration form for cancer diagnosis or treatment. In the case of cancer treatment, they can be administered in a pharmaceutically effective amount to treat cancer cells or their metastasis, or to inhibit cancer growth. The type of cancer, the age and weight of the patient, the nature and severity of symptoms, the type of current treatment, the number of treatments, the route and route of administration, and can be readily determined by those skilled in the art. The composition of the present invention may be administered together with the above-described pharmacological or physiological components or may be administered sequentially, or may be administered in combination with additional conventional therapeutic agents, and administered sequentially or concurrently with conventional therapeutic agents. Such administration may be single or multiple administrations. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

In the present invention, 'administering' means introducing a predetermined substance into a subject by any suitable method, so that the administration route of the composition comprising the antibody of the present invention is administered through any conventional route so long as it can reach the target tissue . But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. However, at the time of oral administration, since the protein is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top. In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Expression and purification of fusion protein of cancer cell specific antibody and ZR peptide

In the present invention, a HER2-specific antibody known to be overexpressed in breast cancer cells was used to produce a cancer cell-specific antibody and a fusion protein of ZR peptide, and a 4D5 antibody and a ZR peptide (Fab fragments, Herceptin antibody) LEI RAAFLRR RNTALRT RVAELRQ RVQRLRN IVSQYET RYGPL; SEQ ID NO: 2-Bioneer, Korea).

The 4D5Fab antibody-ZR peptide gene and the pKB-Fab100 vector were treated for 1 hour and 30 minutes at 37 ° C using Not (NEB cat No. r0189s) and purified. The purified pKB-Fab100 vector and ZR peptide 20 μl of 10 x ligase buffer solution, 10 μl of T4 DNA ligase and sterile distilled water were added to make a total volume of 200 μl, and then 16 Overnight at 37 ° C and ligation was performed to prepare a pKB-Fab100 vector containing the ZR peptide gene.

Thereafter, the cells were transfected into E. coli TG1 cells (ATCC 39078) by electroporation (using BioRad Gene Pulser Xcell) under the conditions of 2.5 kV, 200 O, and 25 D to prepare 4D5 Fab antibody-ZR A recombinant microorganism having the ability to produce a fusion protein was prepared.

TG1 cells having the ability to produce the 4D5 Fab antibody-ZR fusion protein were cultured in a 2YT medium (16 g of Bactotryptone, 10 g of yeast extract, 5 g of NaCl and 1 liter of H2O containing ampicillin (100 g / ml) , pH 7.0) and cultured at 37 ° C. to select a single colony. The selected colonies were cultured at 37 ° C. and 200 rpm in 4 L of 2YT ampicillin medium to have an OD ₆₀₀ value of 0.6 to 0.7 . When the OD ₆₀₀ value reached 0.6 to 0.7, 100 μM IPTG (isopropyl-β-D-thiogalactopyranoside) was added and cultured at 26 ° C. and 200 rpm for 16 hours. The cells induced by IPTG were centrifuged at 4000 rpm for 15 minutes and the supernatant was discarded and the remaining cell pellet was resuspended in 40 ml of TES buffer (0.2 M Tris-HCl, pH 8.0; 0.5 mM EDTA ; 0.5 M sucrose) and 2.5 ml of lysozyme (10 mg / ml in TES buffer, SIGMA-ADRICH L6876) was added to the suspended cells. After addition of lysozyme, 250 ml of TES buffer diluted 1: 2 with cold distilled water (about 1 to 4 ° C) was added, and an osmotic shock was applied on ice for 30 minutes. (Hanil, supra 22k) for one minute.

Peripheral cellular extraction (Bothmann et al., Nat Biotechnol. , 16 (4): 376, 1998) was used to purify the 4D5 Fab antibody-ZR fusion protein and as shown in Figure 3, 4D5Fab It was confirmed that the antibody-ZR fusion protein was expressed.

 ZE peptide-immobilized nanoemulsion preparation

A nanoemulsion immobilized with the ZE peptide of the present invention (LEI EAAFLEQ ENTALET EVAELEQ EVQRLEN IVSQYET RYGPL; SEQ ID NO: 3) was prepared, and a basic dye Rhodamin was bound to confirm the degree of binding with the fusion protein for cancer diagnosis .

(Phosphatidylcholine; Egg PC, Avanti Polar Lipids Inc., USA), 20 mol% cholesterol (Sigma-Aldrich Co., USA), 3 mol% DSPE-mPEG2000 -glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) 2000; Avanti Polar Lipids Inc., USA), 1 mol% DPPE-rhodamine (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N - ( lissamine rhodamine B sulphonyl; Avanti Polar Lipids Inc., USA), 2.6 mol% DSPE-PEG3400- ℃ NH 2 (N- (aminopropylpolyethyleneglycol) carbamyl-distearoylphosphatidyl ethanolamine) were dissolved in chloroform, and the chloroform was evaporated under a reduced pressure device, The complex in which chloroform was evaporated was dried in a vacuum oven at 50 캜 for one day to form a thin lipid membrane.

Then, sterilized third distilled water was added to the membrane and dispersed by ultrasonic waves for 10 minutes to prepare a liposome suspension. To the suspension thus prepared, 2.0% w / v emulsifier solution and 40% v / v perfluorocarbon were added , Followed by mixing for 4 minutes with a homogenizer (Fisher Scientific, USA). The mixed solution was suspended at 20000 psi for 4 minutes using a microfluidizer (M-110s Microfluidizer, microfluidics Co., USA), and the prepared nanoemulsion was desalted using a desalting column (GE Healthcare, UK) The residue was removed and refrigerated at 4 ° C.

A ZE peptide (20 mg / ml) having a carboxyl group was activated by using a 10 mM EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) solution to bind the ZE peptide to the prepared nano- The mixture was mixed with the prepared nanoemulsion at room temperature for 2 hours to covalently bond the two substances. Finally, a nanoemulsion (ZE-Rhodamin-PFCE) having a surface-immobilized ZE peptide was prepared.

Identification of nanoemulsion properties and cytotoxicity of ZE peptide immobilized on the surface

3-1: Characterization

In order to confirm the characteristics of the ZE-Rhodamin-PFCE prepared in Example 2, the emission peak, the diameter and the polydispersity (the property that the particle size of the dispersion system is not uniform) were measured And a nanoemulsion in which the ZE peptide was not immobilized was used as a control.

UV-vis and fluorescence spectra were measured to investigate the fluorescence properties of the nanoemulsion. The UV-vis spectrum (Beckman Coulter, USA) was measured in the range of 400 to 700 nm. The fluorescence spectrum (LS55 fluorescence spectrometer, PerkinElmer, USA) nm and an emission wavelength of 600 nm, respectively.

The diameter and the polydispersity of the nanoemulsion were measured by using DLS (Dynamic Light Scattering, Otsuka, Japan). As a result, it was confirmed that the diameter of the nanoemulsion was 80 to 180 nm, the polydispersity was 0.087, It was confirmed that the ZE peptide had a value similar to that of the non-immobilized nanoemulsion (FIGS. 4A and 4C).

As a result, it was confirmed that the emission wavelength peak of the nanoemulsion in which the ZE peptide was immobilized was 592 nm, the diameter was 123.1 ± 26.9 nm, the polydispersity was 0.087, and that the ZE peptide had a value similar to that of the nanoemulsion without immobilization (Figs. 4A and 4C).

3-2: Cytotoxicity check

To determine the cytotoxicity of ZE-Rhodamin-PFCE prepared in Example 2, nanoemulsion was added to SKB3 and MDF7, which are breast cancer cell lines, and MCF10A, which is a general breast cell, by concentration (0.032 μL / mL, 0.16 μL / mL, 0.8 mL / mL, 4 μL / mL, 20 μL / mL and 100 μL / mL), respectively, and cell viability was measured by MTT assay.

To examine the cytotoxicity of the prepared nanoemulsion, each cell was grown in a 96-well plate. To stop the cell proliferation, samples were diluted to the concentration in serum-free medium, and 100 μL was added to each cell-grown well. The cells were cultured in a 37 ° C, 5% CO2 incubator for 24 hours and 48 hours The surviving water of each well to which samples were added by the following MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) (80% Cytotoxic Concentration). The concentration of the sample in which 80% of the cells survived was determined by CC80 (80% Cytotoxic Concentration). The MTT detection method reduces the mitochondrial dehydrogenase in surviving cells to yellow for MTT and formazan in purple, dissolves the product in organic solvent, and measures the number of living cells and dead cells Respectively.

As a result, it was confirmed that SKBR3, MDF7, and MCF10A showed a survival rate of about 100% regardless of the concentration of the nanoemulsion, and it was confirmed that the nanoemulsion of the present invention was not toxic to cells (FIGS. 4B and 4C).

Measurement of Efficacy of Cancer Diagnostic Fusion Protein Fused with ZR Peptide and Antibody Specific to Cancer Cell Surface Receptor and Cancer Therapeutic Nano Emulsion Fixed to Surface of ZE Peptide

ZE-Rhodamin-PFCE was added to the target cell-4D5 Fab-ZR fusion protein complex to confirm that the 4D5 Fab antibody-ZR fusion protein prepared in the present invention was targeted to HER2 overexpressed cells (SKBR3) The binding (ZR-ZE) was confirmed by flow cytometry (FACS) experiments.

As a control group of the fusion protein, a fusion protein in which a Fab fragment (SIGMA, IgG Fab) other than a HER2-specific antibody was fused with a ZE peptide was prepared and used in the same manner as in Example 1. As a control group of cells, MCF7, a breast cancer cell line, was used.

As a result, the 4D5Fab-ZR fusion protein selectively binds to the SKBR3 cell line overexpressing (FITC labeling) Her2, and ZE-Rhodamin-PFCE forms a zipper pair with the ZR peptide to bind to the target cell-4D5Fab-ZR fusion protein complex (Fig. 5).

In addition, MCF10A and SKBR3 cells, which are normal breast cells, were cultured and treated with 4D5Fab antibody-ZR fusion protein and ZE-Rhodamin-PFCE, and then observed under a microscope.

For the 4D5Fab antibody-ZR fusion protein and ZE-Rhodamin-PFCE imaging, each cell was divided into 8 well plates at a concentration of 2 * 10 ⁴ cells / well, and 10 ug / ml 4D5 Fab antibody- Treatment After washing, 10 ug / ml ZE-Rhodamin-PFCE was treated for 1 hour and the cells were washed and fixed to measure fluorescence imaging.

As a result, it was confirmed that 4D5Fab-ZR fusion protein binds only to SKBR3 cells, and ZE-Rhodamin-PFCE also binds ZR peptide and zipper pair to bind to target cell-4D5Fab-ZR fusion protein complex ).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Composition for Cancer Diagnosis and Therapy Using Leucine Zipper          Pair <130> P14-B089 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> HER-2 specific 4D5 <400> 1 Thr Val Ala Gln Ala Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly   1 5 10 15 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly              20 25 30 Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly          35 40 45 Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr      50 55 60 Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr  65 70 75 80 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                  85 90 95 Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Asp Gly Phe Tyr Ala             100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly         115 120 125 Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Asp Ile Gln Met     130 135 140 Thr Gln Ser Ser Ser Ser Ser Ser Val Ser Ser Val Gly Asp Arg Val Thr 145 150 155 160 Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr                 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser             180 185 190 Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly Ser Ser Ser Gly         195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala     210 215 220 Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly                 245 250 255 Ser Gly Gly Gly Gly Ser Gly Gln Ala Gly Gln His Ala Ala             260 265 270 <210> 2 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> ZR peptide <400> 2 Leu Glu Ile Arg Ala Ala Phe Leu Arg Arg Arg Asn Thr Ala Leu Arg   1 5 10 15 Thr Arg Val Ala Glu Leu Arg Gln Arg Val Gln Arg Leu Arg Asn Ile              20 25 30 Val Ser Gln Tyr Glu Thr Arg Tyr Gly Pro Leu          35 40 <210> 3 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> ZE peptide <400> 3 Leu Glu Ile Glu Ala Ala Leu Glu Glu Glu Asn Thr Ala Leu Glu   1 5 10 15 Thr Glu Val Ala Glu Leu Glu Glu Glu Val Gln Arg Leu Glu Asn Ile              20 25 30 Val Ser Gln Tyr Glu Thr Arg Tyr Gly Pro Leu          35 40

Claims (7)

A fusion protein for breast cancer diagnosis in which Herceptin or 4D5 Fab antibody and ZR peptide are specifically fused to HER2 (human epidermal growth factor receptor 2 protein); And a cancer therapeutic nanoemulsion containing a gene or drug having an anticancer activity immobilized thereon, wherein ZE peptide is immobilized on the surface, and which can selectively treat breast cancer after diagnosis of breast cancer.
2. The composition for diagnosing and treating breast cancer according to claim 1, wherein the ZR peptide is represented by the amino acid sequence of SEQ ID NO: 2 and the ZE peptide is represented by the amino acid sequence of SEQ ID NO:
delete delete The composition for diagnosing and treating breast cancer according to claim 1, wherein the fusion protein for breast cancer diagnosis or the cancer therapeutic nanoemulsion is further bound with a fluorescent substance.
The method of claim 5, wherein the fluorescent material is at least one selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine ), FITC (Fluorescein isothiocyanate), Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC (Tetramethyl Rhodamine Iso-Thiocyanate) , A cyanine dye, and a thiadicarbocyanine dye. The composition for diagnosing and treating breast cancer according to claim 1, wherein the dyes are selected from the group consisting of dyes, cyanine dyes, and thiadicarbocyanine dyes.
The nanoemulsion according to claim 1, wherein the nanoemulsion is selected from the group consisting of (a) phosphatidylcholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine N- [methoxy polyethylene glycol, DPPE-rhodamine (2-dioleoyl- sn -glycero-3-phosphoethanolamine- N - (lissamine rhodamine B sulphonyl) and DSPE-PEG3400-NH ₂ (aminopropylpolyethyleneglycol) carbamyl-distearoylphosphatidyl ethanolamine) were dissolved in chloroform and chloroform (B) preparing a liposome suspension by dispersing the lipid membrane with distilled water and ultrasonically dispersing the lipid membrane, (c) mixing the emulsion solution and perfluorocarbon in the suspension to form a nano emulsion And (d) covalently binding a ZE peptide to the surface of the nanoemulsion. [Claim 10] The composition for diagnosing and treating breast cancer according to claim 1,

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