KR101253709B1 - Secretory fusion protein for targeting and tracing - Google Patents

Abstract

The present invention relates to a secretory target tracking fusion protein, more specifically secretory target tracking fusion consisting of a peptide, a molecular imaging reporter or therapeutic peptide or protein having an extracellular secretion function and a peptide having a specific binding ability to the target cell It is about protein.
According to the present invention can be prepared secreted target tracking fusion protein consisting of a peptide, a molecular image reporter or therapeutic peptide or protein having an extracellular secretion function and a peptide having a specific binding ability to the target cell, the fusion protein is a target Specific binding to the cells, can be confirmed by imaging the binding. In addition, the induction promoter can be used to express the fusion protein only when necessary. In addition, the therapeutic peptide or protein can be used to search for therapeutic agents that can treat target cells. Therefore, the secretion-type target tracking fusion protein of the present invention may be usefully used as a molecular imaging agent or a tumor cell therapeutic agent.

Description

Secretion fusion protein for targeting and tracing}

The present invention relates to a secretory target tracking fusion protein, more specifically secretory target tracking fusion consisting of a peptide, a molecular imaging reporter or therapeutic peptide or protein having an extracellular secretion function and a peptide having a specific binding ability to the target cell It is about protein.

Attempts have been made in the diagnosis or treatment of diseases to ensure that the target substance selectively stays and acts only on the target tissue or cell. In particular, many studies have been conducted on the proteins that are specifically present in target cells or tissues in the treatment of tumors, the diagnosis and treatment of osteoarthritis and brain diseases. . For example, in prostate cancer, many prostate specific antigens (PSAs) are present, and in arthritis and various tumor tissues, matrix metalloprotease (MMP) is specifically expressed higher than that of normal tissues. These are being used as targets for disease research and treatment. However, if a substance used for the diagnosis and treatment of a disease does not act specifically on such a target, side effects due to a non-specific distribution or a problem of low image quality may occur. Therefore, it is necessary to develop a formulation that acts only on a target. It is becoming.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior arts, and as a result, minimize the problems of side effects or low image quality due to the nonspecific distribution of the existing disease diagnosis or treatment, and the material for diagnosis or disease treatment In order to specifically deliver to the target cells, a secretory target fusion protein was prepared, which consists of a peptide, a molecular imaging reporter or therapeutic peptide or protein having an extracellular secretion function, and a peptide having a specific binding ability to the target cell. It was confirmed that the protein specifically binds to the target cell and effectively images it, thus completing the present invention.

It is therefore a primary object of the present invention to provide a secreted target tracking fusion protein with extracellular secretory function, imaging or therapeutic function and target function.

Another object of the present invention is to provide a molecular imaging diagnostic agent or tumor cell, infection or inflammatory disease, ischemic disease treatment using the secreted target tracking fusion protein.

According to an aspect of the present invention, the present invention provides a secretory target tracking fusion protein consisting of a peptide, a molecular imaging reporter or therapeutic peptide or protein having an extracellular secretion function and a peptide having a specific binding ability to a target cell. A schematic of the secreted target tracer fusion protein is shown in FIG. 1.

The fusion protein of the present invention is characterized by having multifunctionality. The term 'multifunctional' means that, for example, when the secretory target tracking fusion protein of the present invention is Gluc-mCherry-RGD, Gluc generates a bioluminescence image signal, mCherry generates a fluorescent signal, and RGD is an integrin Since the fusion protein has three functions, and the mCherry portion of the fusion protein is replaced with TRAIL having a therapeutic effect, TRAIL has the effect of a cell therapy. In this case, since RGD has a function of tracking integrin expressed in tumor cells or tumor blood vessels, the secreted target tracking fusion protein of the present invention has three or more multifunctional functions.

In the fusion protein of the present invention, the peptide having an extracellular secretory function is secreted gaussia luciferase (Sec-Gluc), secretory antibody, or signal recognition particle (signal recognition particle) Characterized in that it is a peptide having an extracellular secretory function selected from the group consisting of a peptide comprising a hydrophobic amino acid sequence recognized by, but is not limited thereto. More specifically, the peptide having the extracellular secretory function is preferably secreted type Gaussian luciferase (Sec-Gluc) having the amino acid sequence of SEQ ID NO: 3.

The extracellular secretory peptide has a function of allowing extracellular secretion of a molecular image reporter or therapeutic peptide or protein and a peptide having a specific binding ability to a target cell in cells or bacteria.

In the fusion protein of the present invention, the molecular image reporter is a protein to which a fluorescent protein, a bioluminescent protein, a photoacoustic protein, a radionuclide and a radiocontrast agent, an ultrasound contrast agent or an MR contrast agent bind. Molecular imaging reporter selected from the group consisting of, the therapeutic peptide or protein is TRAIL, apoptin, cytolysin (cytolysin), INF-β, FASL, TNF-α, cytokines (IL-2 or IL-18), blood vessels Anti-angiogenic peptides (thrombospondin, endostatin), ischemic peptides (pro-angiogenic peptide-vascular endothelial growth factor, hepatocyte growth factor, angiopoietin, placental growth factor) or defensin and cathelicidin It is characterized in that the therapeutic peptide or protein selected from the group consisting of antimicrobial peptides, such as It is not. More specifically, the molecular image reporter is preferably a fluorescent protein mCherry having an amino acid sequence of SEQ ID NO: 4, the therapeutic peptide is preferably TRAIL having an amino acid sequence of SEQ ID NO: 6.

In addition, the molecular image reporter or therapeutic peptide is characterized in that the peptide having a binding capacity with the molecular image reporter or therapeutic material. The substance includes a molecular imaging reporter or a therapeutic peptide or protein, as well as a therapeutic compound biotinylated material (biotinlylated polyamine dendrimer carrying drug, biotinlylated geldanamycin), a diagnostic compound biotinylated material (biotinlyated lipids with Tc-99m HMPAO), avidinlyated It may also be saporin or avidinylated micorbubble, which may be used for treatment and diagnosis.

For example, when a biotin acceptor peptide (eg, GLNDIFEAQKIEWHE) is introduced into the fusion protein of the present invention, binding to a biotinylated material is possible, or streptavidin labeling binding may be used after biotin binding. In other words, if biotin or streptavidin is labeled with a diagnostic and therapeutic material, it can be used for diagnosis and treatment.

In addition, the peptide having a specific binding capacity to the target cells are RGD, tumor angiogenesis targeting (NGR), apopep-1 (apoptosis targeting peptide), GPC-3 (glypican-3) target peptide (liver cancer, gonadal cancer, melanoma) GPC-3 tracer peptide overexpressed), IL-12 target peptide, aminopeptidase P target peptide, IL11-γ target peptide, Nucleolin target peptide, tumor lymphatic target peptide Characterized in that the peptide is selected from the group, it is not limited thereto. Table 1 shows the peptide sequences having specific binding ability to target cells [Liu et al., Pat . Anticancer . Drug . Discov . Nov; 3 (3): 202-8, 2008]. More specifically, the peptide having a specific binding ability to the target cell is preferably RGD having an amino acid sequence of SEQ ID NO: 5.

Table 1. Peptides with Specific Binding Ability to Target Cells]

The secretory target tracking fusion protein of the present invention is secreted extracellularly to track and bind to the target cells by a peptide having a specific binding ability to the target cells, and imaged by a molecular imaging reporter, or through a therapeutic peptide or protein Target cells can be treated by mechanisms that further accelerate cell death.

In order to release a specific protein or peptide site from a tumor site, the fusion protein of the present invention is a peptide that causes extracellular secretion of the fusion protein, for example, a therapeutic peptide connection site, a therapeutic peptide, between each protein or peptide constituting the fusion protein. A peptide sequence that may be cleaved by MMP-2 or MMP-9 may be further included at the connection portion of the peptide and the cell tracer peptide. Preferably, the peptide sequence that can be cleaved by MMP-2 or MMP-9 is PLGLAG. By further including a peptide sequence that can be cleaved by the MMP in the fusion protein of the present invention, it is possible to separate the peptide having a cell therapeutic effect such as TRAIL only at the tumor site.

The secreted target tracking fusion protein of the present invention is preferably a peptide having an extracellular secretory function of SEQ ID NO: 3, a molecular image reporter or therapeutic peptide or protein of SEQ ID NO: 4, and specific binding ability to a target cell of SEQ ID NO: 5 It is characterized by consisting of a peptide having.

The fusion protein of the present invention is characterized in that the αvβ3 target tracking fusion protein having the amino acid sequence of SEQ ID NO: 2.

Integrins are heterodimers in which α and β subunits are covalently linked to each other, and the β1 subfamily has been known as a major mediator of interstitial adhesions and may have other functions such as direct mediation of cell-cell adhesions. There is a research report showing that [Larjava et al., J. Cell . Biol . 110: 803-815, 1990. The β2 subfamily found in leukocytes contains receptors that mediate cell-cell interactions. The β3 subfamily contains the platelet glycoprotein IIb / IIIa complex and the vitronectin receptor, and has the potential to play an important role in tumor invasion and development of malignant tumors [Albelda et al., Cancer . Res . 50: 6757-6764, 1990.

The integrin αvβ3 has been reported to increase its expression upon neovascularization, which is essential for the growth of cancer cells, and has potential as a target for anticancer drug carriers. Integrin Receptor Interactions with Ligands For example, 3,3'5,5'-tetraiodothyroacetic acid (Tetrac), which blocks the interaction of integrin αvβ3 with its ligand, is an integrin αvβ3 antagonist that prevents the spread of tumor cells by integrin. . In addition, by inhibiting the signaling of integrins expressed on the surface of cancer cells, they induce cell death and cause cancer cell death [Yalcin et al., Anticancer . Res . 10, 3825-31, 2009].

Integrin αvβ3 is not only expressed in neovascularization of the tumor site but is also overexpressed in ischemic tissues such as myocardial infarction limb ischemia, myocardial remodeling, ocular retinopathy and joint and skin inflammatory tissues. Integrin αvβ3 expression can also be used for diagnostic / condition assessment / and therapeutic delivery [Lee et al., J. Korean . Med . Assoc . 52 (2): 135-142 (2009); Zhou et al., Theranotics . 1: 58-82 (2011).

According to another aspect of the present invention, a gene encoding the secreted target tracking fusion protein of the present invention is provided.

In the present invention, the gene is characterized by having the nucleotide sequence of SEQ ID NO: 1.

The gene is characterized in that it further comprises an inducible promoter (inducible promoter). The secreted target tracking fusion protein of the present invention can be used to express the fusion protein only when necessary using an inducible promoter. For example, using a promoter capable of inducing or stopping expression by tetracycline, expression can be controlled by administering or eliminating tetracycline only when the production of the secreted target tracer fusion protein is necessary [ Tetracycline (Tet) inducible expression system; Tet-off and Tet-on system. This inducible promoter system is considered to be very useful when applied to the gene therapy technique of the secreted target tracking fusion protein of the present invention in a transgenic animal model or cell therapy technology.

According to another aspect of the invention, the invention provides a recombinant vector comprising a gene encoding said secreted target tracking fusion protein.

In the present invention, any expression vector capable of expressing the gene may be used as the recombinant vector expressing the gene, and preferably, pcDNA3.1, pRSET (B), and pcDNA3.0 may be used. Since the pRSET (B) vector is expressed in JM109 bacteria and the pcDNA vector is expressed in mammalian cells, the pRSET (B) vector can be used to obtain a large amount of secreted target tracking fusion protein that can be traced to target cells.

In the present invention, the recombinant vector is a pcDNA3.0-sGluc-mCherry-RGD × 3 vector having a cleavage map of FIG. 3.

According to another aspect of the present invention, the present invention provides a cell line transformed with the recombinant vector.

In the present invention, the cell line may be mammalian cells, bacteria or yeast, more specifically, the cell line is characterized in that the Chinese hamster ovary cell (CHO), but is not limited thereto. In the present invention, after confirming mRNA expression of a target molecule such as αvβ3 in breast cancer cell line (MDM-MB-231), lung cancer cell line (A549), human synovial cell lines (2046 and 2047) and CHO, CHO which does not express αvβ3 is identified. It was used as a transformed cell line.

The transformed cell line is characterized in that the CHO-sGluc-mCherry-RGD.

In a specific embodiment of the present invention, the transformed cell line CHO-sGluc-mCherry-RGD is introduced by using a liposome (lipofectamine) as a recombinant vector pcDNA3.0-sGluc-mCherry-RGF × 3, which is produced in CHO cells. Produced. In addition, when culturing the transformed cell line and cells, it was confirmed that the fusion protein of the present invention is expressed in the culture medium and cell lysate (see Example 3-2 and Figure 7).

In the present invention, the transformed cell line may be transformed using protoplast, electroporation, or viral gene transfer, but in the present invention, pcDNA3.0-sGluc-mCherry-RGD Transformed cell lines were constructed by importing the x3 gene using liposomes and selecting the cells into which the gene was introduced using antibiotics and FACS sorter.

In addition, a transgenic animal model transformed with the recombinant vector can be prepared. The transgenic animal model may produce a transgenic mouse using a mouse, and the expression level of the target cell may be continuously monitored by the secretion imaging reporter of the secreted target tracking fusion protein of the present invention expressed in the transgenic mouse. Can be. For example, constructing a transgenic mouse that expresses a fusion protein of an RGD peptide and a secretion imaging reporter that tracks αvβ expressed in neovascularized blood vessels allows continuous non-invasive imaging of neovascularization, neovascularization or tumors. It can be used as a search animal model for substances that inhibit angiogenesis. When neovascularization occurs by implanting a tumor into the animal model, the secretory imaging reporter secreted from the cells of the animal model is localized at the tumor site to allow imaging and to reduce the neovascularization of the tumor tissue. Can be screened.

According to an aspect of the present invention, the present invention provides a molecular imaging diagnostic agent containing the secreted target tracking fusion protein as an active ingredient.

In the molecular imaging diagnostic agent of the present invention, the secretory target tracking fusion protein is characterized by having an amino acid sequence of SEQ ID NO: 2.

According to an aspect of the present invention, the present invention provides a tumor cell therapeutic agent containing the secreted target tracking fusion protein as an active ingredient.

Since the therapeutic peptide (eg, TRAIL) of the secreted target tracking fusion protein is known to be effective in treating tumor cells, [Argiris et al., Exp . Biol . Med . ( Maywood ) . May 1; 236 (5): 524-36 (2011)], the secreted target tracer fusion proteins can be used as tumor cell therapeutics.

The present invention provides an agent for treating an infectious or inflammatory disease containing the secreted target tracking fusion protein according to the present invention as an active ingredient.

Therapeutic peptides (eg, defensins, cathelicidin LL-37, MIP-3α / CCL20, buforin I or ubiquicidin) of the fusion protein are infected or inflammatory. Since it is known to be effective in treating diseases, Wiesner et al., Virulence . Sep-Oct; 1 (5): 440-64 (2010)], the secreted target tracer fusion proteins can be used as therapeutic agents for infectious or inflammatory diseases.

The present invention also provides a therapeutic agent for ischemic disease containing the secreted target tracking fusion protein of the present invention as an active ingredient.

Since the therapeutic peptide (eg, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), angiopoietin or PLGF (placental growth factor)) of the fusion protein is known to be effective in treating ischemic disease, Beohar et al., J. Am . Coll . Cardiol . Oct 12; 56 (16): 1287-97 (2010); Mac et al., Wiley . Interdiscip . Rev. Syst . Biol . Med . Nov-Dec; 2 (6): 694-707 (2010); Taniyama et al., Nippon Rinsho . May; 68 (5): 949-52 (2010)], the secreted target tracking fusion protein can be used as a therapeutic agent for ischemic disease.

Tumor cell therapy, infection or inflammatory disease treatment or ischemic disease treatment agent of the present invention can be prepared by a method known in the pharmaceutical art for use as a therapeutic agent, a pharmaceutically acceptable carrier, excipient, diluent, etc. The mixture may be prepared and used in the form of powder, granules, tablets, capsules, or injections. They can also be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically) or orally.

The therapeutic agents of the invention are administered in a therapeutically effective amount. The term “therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the patient's age, sex, weight, health condition and symptoms of the disease. It can be appropriately selected depending on the administration time, administration method, preferably 0.01 ~ 100 mg per day of adult can be administered.

As described above, according to the present invention, a secretory target tracking fusion protein consisting of a peptide having a function of extracellular secretion, a molecular image reporter or therapeutic peptide or a protein, and a peptide having a specific binding ability to a target cell may be obtained. The fusion protein may specifically bind to target cells, and may be confirmed by imaging the binding. In addition, the induction promoter can be used to express the fusion protein only when necessary. In addition, it is possible to apply to cell therapy by building an immune cell line capable of producing the fusion protein. In addition, the present invention can search for a therapeutic agent that can treat a target cell using a therapeutic peptide or protein. Therefore, the secretory target tracking fusion protein of the present invention can be usefully used as a molecular imaging agent or a tumor cell therapeutic agent, and can be used for the search for a therapeutic agent.

In addition, the diagnosis of neovascularization such as ischemic diseases, retinopathy, and inflammatory diseases including myocardial infarction and lower limb ischemia other than tumors can be used as a diagnostic method for molecular imaging and treatment of diseases related to the development and progression of the disease. It is considered possible.

Figure 1 shows a schematic of the secreted target trace fusion protein of the present invention.
Figure 2 is a schematic diagram showing the construction of pcDNA3.0-sGlu-mCherry-RGD × 3 recombinant vector.
3 shows a cleavage map of the recombinant vector.
4 shows the results of measuring αvβ3 expression in various cell lines.
Figure 5 is the result of measuring the protein expression in culture medium and cell lysate when incubating the fusion protein of the present invention with bacteria.
6 is a result of observing whether the recombinant vector of FIG. 3 is transformed into a CHO cell line and then introduced into cells by mCherry fluorescence.
Figure 7 is a result of measuring the protein expression in culture medium and cell lysate when culturing the fusion protein of the present invention with mammalian cells (CHO).
8 is a result of measuring whether the fusion protein of the present invention specifically binds to αvβ3 in CHO and synovium cells using a luminescent enzyme (Galusssia luciferase).
9 is a result of measuring whether the fusion protein of the present invention specifically binds to αvβ3 in CHO and synovium cells using mCherry fluorescent protein.
FIG. 10 is an image showing sGlu-mCherry-RGD × 3 secreted in U87MG-EGFP cells expressing αvβ3 and showing fluorescence signals. FIG.
11 is an image showing that sGlu-mCherry without RGD having a target tracking function did not show fluorescence signal in U87MG-EGFP cells expressing αvβ3.
12 is an image showing that sGlu-mCherry-RGD × 3 secreted in U87MG-EGFP cells expressing αvβ3 is attached to show a fluorescent signal.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1.vector ( Vector Production

Using PCR, amplify the secretory gaussia luciferase gene portion from pcDNA3.1-sGluc vector (Professor Jeong-Jun Min, Stanford University Dr. Paulmurgan), and then insert the amplified gene into the pRSET (B) vector (Invitrogen). (B) -sGlu (secretory Gluc) vectors were constructed. Thereafter, RGD (Arg-Gly-Asp) nucleotide sequence was repeated three times the primer binding (annealing) to prepare a complementary strand RGD sequence (RGD × 3) was prepared. In addition, only mCherry gene was amplified in the pmR-mCherry vector (Clonetech) using PCR, and the amplified gene was inserted into the pRSET (B) vector to prepare a pRSET (B) -mCherry vector. Subsequently, the prepared RGD sequence (RGD × 3) was inserted into the prepared pRSET (B) -mCherry vector to prepare a pRSET (B) -mCherry-RGD × 3 vector. After cutting the mCherry-RGD × 3 gene using the Kpn I and EcoR I restriction enzymes in the newly prepared pRSET (B) -mCherry-RGD × 3 vector, the pRSET (B) -sGlu vector was previously prepared. PRSET (B) -sGlu-mCherry-RGD × 3 vector was prepared. The pRSET (B) -sGlu-mCherry-RGD × 3 vector was sheared into the sGlu-mCherry-RGD × 3 gene using BamHI and EcoRI restriction enzymes, inserted into a pcDNA3.0 vector (Invitrogen), and the final product, pcDNA3. A .0-sGlu-mCherry-RGD × 3 vector was constructed.

The pcDNA3.0-sGlu-mCherry-RGD × 3 vector construction process is shown in FIG. 2, and a cleavage map of the recombinant vector is shown in FIG. 3.

Example  2. αvβ3 Expression in Various Cell Lines

To confirm the expression of αvβ3 in various cell lines, RNA was isolated from breast cancer cell lines (MDA-MB-231), lung cancer cell lines (A549), human synovial cell lines (2046 and 2047) and Chinese hamster ovary cells (CHO). First, after culturing each cell in a 100 mm dish, RNA was extracted from the cells using Trizol (Invitrogen), and then synthesized cDNA using the RT-PCR method, and then synthesized cDNA. MRNA expression was confirmed by PCR and DNA electrophoresis using primers (short strands complementary to specific sequences).

As a result, as shown in FIG. 4, the expression of mRNA through RT-PCR was confirmed by the electrophoresis of αv and β3 in all cells except CHO cells, but CHO was not expressed by electrophoresis on both αv and β3. Did.

Example  3. Fusion Protein Expression Analysis

Since the pRSET vector is expressed in JM109 bacteria, it can be used to obtain a large amount of secreted target tracking fusion protein that can be traced to target cells.

Since the pcDNA vector is expressed in mammalian cells, it can be used to obtain a large amount of secreted target tracking fusion protein capable of tracking target cells, and is also a vector that can be used for cell-based treatment.

3-1. In bacterial culture

Bacteria that can produce the secreted target trace fusion protein of the present invention were produced and cultured together to confirm their release into the culture. First, the pcDNA3.0-sGlu-mCherry-RGD × 3 vector prepared in Example 1 was transformed into competent cells (DH5α), then 50 μg / ml of ampicillin was added and only 2 ml were taken to shake at 37 ° C. Incubate overnight in incubator. After incubation, only 100 μl in 2 ml was taken and centrifuged at 1500 rpm for 5 minutes, after which the supernatant was transferred to a new tube. The supernatant was removed and 100 μl of cell lysis buffer (5 × lysis buffer, promega) was added to the remaining precipitated cell pellet and voltex. After centrifugation again at 1500 rpm for 2 minutes, 20 μl of the supernatant was placed in a 96-well luminometer plate. Thereafter, 100 μl of the gaussia luciferase substrate was added to the plate, and confirmed by a luminometer.

As shown in FIG. 5, the activity of the cell culture medium and the intracellular luciferase was 8 times higher in the luciferase activity in the cell culture medium than the intracellular luciferase activity.

In addition, when the pRSET (B) -sGlu-mCherry-RGD × 3 vector is introduced into the JM109 bacterium, the fusion protein can be obtained in the same manner in culture and bacteria.

3-2. Mammalian Cell Culture

Mammalian cells were produced that could produce the secreted target tracking fusion protein of the present invention and cultured together to confirm their release into the culture. First, in order to construct a stable cell line expressing CHO-sGluc-mCherry-RGD, the pcDNA3.0-sGlu-mCherry-RGD × 3 vector prepared in Example 1 on CHO cells without αvβ3 expression The cells were introduced into cells using liposomes (Lipofectamine, Invitrogen), and then passaged and cultured using only mCherry fluorescence with flow cytometry to classify only cells into which cells were safely introduced with pcDNA3.0-sGlu-mCherry-RGD × 3 (Fig. 6). Thereafter, the CHO-sGluc-mCherry-RGD prepared above was inoculated with a CHO cell line stably immobilized with a control CHO cell which is a parental cell to 5 × 10 ⁴ / well in a 24-well plate. 24 hours after cell inoculation, the media of the wells were transferred to a new tube and the cells in the plate were washed twice with PBS. Then, 100 μl of cell lysis buffer (5 × lysis buffer, promega) was added to lyse the cells at the bottom of the plate. Lysed cells were placed in fresh E-tubes and centrifuged for 2 minutes at 1500 rpm. 20 μl each of the supernatant was added to a 96-well luminometer plate, and 20 μl of the medium added to the tube was added to the plate. Then, 100 μl of the gaussia luciferase substrate was added thereto, and checked by a luminometer.

As a result, as shown in Figure 7, the activity of the cell culture medium and intracellular luciferase lucifer of CHO cells stably immobilized with CHO-sGluc-mCherry-RGD compared to the parental CHO cells without any genes. The lyase activity was 30 times higher and the luciferase activity in cell culture medium was 500 times higher.

Example  4. Confirmation of αvβ3 Specific Binding of Fusion Proteins

4-1. Luminase ( Glusssia luciferase ) Use

After inoculating CHO and synovium cells in 5 × 10 ⁴ / well in 6-well plates, the culture medium obtained during the incubation of CHO-sGluc-mCherry-RGD the next day was 2 ml / put well. After incubation for 48 hours, it was washed twice with PBS. Thereafter, PBS was added to each well at 1 ml / well, coelentrazine (0.25 μg in 50 μl PBS) was added, and an IVIS image was obtained (FIG. 8A). At this time, the reason for using the luminescent enzyme, it is difficult to image the signal of mCherry fluorescent protein with the IVIS imaging equipment, the luminescent enzyme was used because the sensitivity is higher than the fluorescent protein. In a live animal, a high sensitivity video signal is required to evaluate the video signal, but the sensitivity of the light signal is higher than that of mCherry.

As shown in FIG. 8, synovial cells 2052 expressing αvβ3 showed a 20-fold increase in luminescence image signal compared to CHO cells not expressing αvβ3 (FIG. 8B). . Therefore, it can be seen that the fusion protein specifically binds to αvβ3.

4-2. Fluorescent protein ( mCherry fluorescent protein ) Use

After inoculating CHO, CHO-sGluc-mCherry-RGD, and synovium cells (2052 cells) on Nunc (Lab Tek-Chamber slide), respectively, CHO + synovium group and CHO-RGD + synovium group were 1 × 10 ⁴ cells, respectively. Mix and inoculate each well. After incubation for 48 hours, the cells were washed twice with warm PBS. Then, 100 μl of BD cytofix / cytoperm 1 × buffer was added to each well and fixed at 4 ° C. for 30 minutes. Then, 1 ml of BD perm / wash buffer and 9 ml of PBS were put into a 15 ml tube to prepare a 1 × Perm / wash buffer. 200 μl of the prepared 1 × Perm / wash buffer was added twice per well and washed twice. Anti-human αvβ3 (milipore, 0.5 μg / 100 μl in 1 × BD Perm / wash buffer) was kept at room temperature for 1 hour, then 200 μl of 1 × BD Perm / wash buffer (buffer for washing antibody at room temperature) per well Was added and washed three times. Secondary antibody FITC Goat-anti mouse (BD, 2 μg / 100 μl in 1 × BD Perm / wash buffer) was left at room temperature for 40 minutes, then 1 × BD Perm / wash buffer (buffer for washing secondary antibody) 200 for each well. μl was added and washed three times. Thereafter, the DAPI was stained by dropping one drop per well, and then the cover glass was covered and mounted. Stained cells were observed by confocal microscopy.

As a result, as shown in Fig. 9, the nucleus of the cell could be observed through DAPI staining, and the mCherry fluorescent protein safely transferred into the cell at the red wavelength of the microscope was confirmed, and the αvβ3-expressing cell was detected through the green filter. I could confirm that. Among them, the secreted protein was found to bind to and express 2052 cells, which could be confirmed through image binding. That is, mCherry fluorescence was observed in the cells expressing αvβ3, indicating that the secretory target tracking fusion protein was bound to the cells expressing αvβ3.

Example  5. Confirmation of αvβ3 Tracking Function of Fusion Proteins

5-1. CHO - sGluc - mCherry - RGD Αvβ3 Tracking of × 3 Fusion Proteins

U87MG cells expressing enhanced GFP in the cytoplasm of U87MG cells expressing αvβ3 expressed in the cytoplasm (U87MG-EGFP) and the fusion protein CHO-sGluc-mCherry-RGD × 3 cells (sGluc-mCherry-) of the present invention. RGD × 3 secreting CHO cells) were mixed in the number of 5 × 10 ⁴ and 1 × 10 ⁵ , respectively, and inoculated in an IWAKI Glass based dish, followed by culture for 48 hours, followed by confocal microscopy.

The microscopic measurement results are shown in FIG. 10. The upper left image (A) is observed as a result of GFP wavelength and U87MG-EGFP cells are observed, and the upper right image (B) is observed as a red wavelength to observe mCherry, and CHO-sGluc-mCherry-RGD × 3 cells are observed. It can be confirmed that the red image signal is observed in the middle of the field of view where U87MG-EGFP cells were observed at the GFP wavelength (bottom image (D)). This demonstrates that the sGluc-mCherry-RGD × 3 secreted target tracking fusion protein secreted from CHO-sGluc-mCherry-RGD × 3 cells tracks U87MG-EGFP cells expressing αvβ3.

5-2. CHO - sGluc - mCherry  Αvβ3 Tracking of Fusion Proteins

U87MG cells expressing enhanced GFP (U87MG-EGFP) and fusion protein CHO-sGluc-mCherry cells (CHO cells secreting sGluc-mCherry), which construct the enhanced GFP in the U87MG cells expressing αvβ3 expressing the EGFP-expressing cell line, respectively. Inoculated in the number of × 10 ⁴ , 1 × 10 ⁵ and inoculated in an IWAKI Glass based dish and cultured for 48 hours, and observed by confocal microscopy (Confocal microscopy).

The microscopic measurement results are shown in FIG. 11. The upper left image (A) was observed with GFP wavelength, and U87MG-EGFP cells were observed, and the upper right image (B) was observed with red wavelength to observe mCherry, and the CHO-sGluc-mCherry cells were observed. have. However, the red image signal was not observed in the middle of the field of view where U87MG-EGFP cells were observed at the GFP wavelength (compare FIG. 10). It can be seen that it is unable to track αvβ3 expressing cells.

These results indicate that the RGD portion of the sGluc-mCherry-RGD × 3 secreted target tracking fusion protein secreted from CHO-sGluc-mCherry-RGD × 3 cells shows the specificity of the target tracking function of αvβ3. The neovascular tracing specificity of the sGluc-mCherry-RGD × 3 fusion protein is shown.

5-3. CHO - sGluc - mCherry - RGD Αvβ3 Tracking of × 3 Fusion Proteins

U87MG cells (U87MG-EGFP) expressing enhanced GFP in the cytoplasm of U87MG cells expressing αvβ3 expressed in the cytoplasm were inoculated in an IWAKI Glass based dish in a number of 5 × 10 ⁴ , and CHO-sGluc-mCherry The culture solution was taken from a dish in which only -RGD × 3 cells were cultured, placed in a dish inoculated with the U87MG cells, and then cultured, and observed with a confocal microscope.

The microscopic measurement results are shown in FIG. 12. The upper left image (A) shows the U87MG-EGFP cells as a result of observation at the GFP wavelength, and the upper right image (B) shows the same as the U87MG-EGFP cells at the GFP wavelength as the result of observing the red wavelength to observe mCherry. It can be seen that a red image signal is observed in the middle of the field of view. This shows that the sGluc-mCherry-RGD × 3 secreted target tracking fusion protein has the ability to track cells expressing αvβ3.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Secretory fusion protein for targeting and tracing <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 1365 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of Secretory fusion protein for targeting and          tracing <400> 1 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgacggtac cggtggtggc atggtgagca agggcgagga ggataacatg 600 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac 660 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag 720 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc 780 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg 840 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg 900 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc 960 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc 1020 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg 1080 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag 1140 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac 1200 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc 1260 atggacgagc tgtacaagct cgagggtggc agcggtggcc gcggcgacgg tggcagcggt 1320 ggccgcggcg acggtggcag cggtggccgc ggcgactaag aattc 1365 <210> 2 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Secretory fusion protein for targeting and          tracing <400> 2 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp Gly Thr Gly Gly Gly Met Val             180 185 190 Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg         195 200 205 Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile     210 215 220 Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys 225 230 235 240 Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu                 245 250 255 Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala             260 265 270 Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp         275 280 285 Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln     290 295 300 Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg 305 310 315 320 Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met                 325 330 335 Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu             340 345 350 Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr         355 360 365 Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu     370 375 380 Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His Asn 385 390 395 400 Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His                 405 410 415 Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Leu Glu Gly Gly Ser Gly             420 425 430 Gly Arg Gly Asp Gly Gly Ser Gly Gly Arg Gly Asp Gly Gly Ser Gly         435 440 445 Gly Arg Gly Asp     450 <210> 3 <211> 185 <212> PRT <213> secretory gaussia luciferase <400> 3 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 4 <211> 234 <212> PRT <213> mCherry <400> 4 Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe   1 5 10 15 Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe              20 25 30 Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr          35 40 45 Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp      50 55 60 Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His  65 70 75 80 Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe                  85 90 95 Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val             100 105 110 Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys         115 120 125 Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys     130 135 140 Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly 145 150 155 160 Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly                 165 170 175 His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val             180 185 190 Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser         195 200 205 His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly     210 215 220 Arg His Ser Thr Gly Gly Met Asp Glu Xaa 225 230 <210> 5 <211> 3 <212> PRT <213> RGD <400> 5 Arg Gly Asp   One <210> 6 <211> 281 <212> PRT <213> TRAIL <400> 6 Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys   1 5 10 15 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala              20 25 30 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys          35 40 45 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr      50 55 60 Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val  65 70 75 80 Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser                  85 90 95 Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro             100 105 110 Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly         115 120 125 Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu     130 135 140 Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly 145 150 155 160 His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile                 165 170 175 His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe             180 185 190 Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln         195 200 205 Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys     210 215 220 Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 225 230 235 240 Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile                 245 250 255 Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala             260 265 270 Ser Phe Phe Gly Ala Phe Leu Val Gly         275 280

Claims (25)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Secreted Gaussian Luciferase (Sec-Gluc), which has an extracellular secretory function and consists of the amino acid sequence of SEQ ID NO: 3,
Fluorescent protein mCherry consisting of the amino acid sequence of SEQ ID NO: 4 as a molecular image reporter, and
A molecular imaging diagnostic agent comprising a secreted target tracking fusion protein consisting of RGD consisting of the amino acid sequence of SEQ ID NO: 5 having specific binding ability to a target cell as an active ingredient.
The diagnostic agent for molecular imaging according to claim 21, wherein the secretory target tracking fusion protein consists of the amino acid sequence of SEQ ID NO: 2. delete delete delete

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