KR101504813B1 - Pharmaceutical composition for immune regulation comprising agonists or antagonists of EGFL8 - Google Patents

Abstract

The present invention relates to an immunomodulatory pharmaceutical composition comprising agonists or antagonists against epidermal growth factor-like protein 8 (EGFL8) and an immunomodulating method using the composition. More particularly, Accordingly, the present invention relates to an immunomodulatory pharmaceutical composition comprising agonists or antagonists of EGFL8 as an active ingredient, and an immunomodulating method using the same, wherein T-cell development (development) when applied to a subject in need of immunomodulation Cell mediated immunity by modulating EGFL8 gene or protein expression or activation of EGFL8 protein identified as a critical negative regulator of T-cell mediated immunity Therefore, it can be used for the treatment of inflammatory diseases such as cancer, rheumatoid arthritis, autoimmune diseases, diabetes, dermatitis, graft-related diseases, Prevention and treatment of immune related diseases such as immune deficiency, as well as can be extensively utilized for such therapeutic development in these areas.

Description

A pharmaceutical composition for immunomodulation comprising an agonist or antagonist of EGFL8,

The present invention relates to an immunomodulating pharmaceutical composition comprising agonists or antagonists against epidermal growth factor-like protein 8 (EGFL8) as an active ingredient, and a method for immunomodulating immune cells using the composition. Enhancing EGFL8 expression or activity when applied to a subject in need of immunomodulation comprising an agonist or antagonist for EGFL8 identified as a negative regulator important for T-cell development Cell mediated immunity (T-cell mediated immunity) by administering an immunomodulating agent to a subject, and a method of immunomodulating immune control using the same.

Thymus is a central lymphoid organ that provides a unique microenvironment for T-cell development and maturation (G. Anderson et al., Semin. Immunol. (2000), 12, 457-464). Thymic epithelial cells (TECs) are a key component of the thymic stroma and promote T-cell differentiation and maturation from lymphoid progenitor cells. In this process, thymic stromal cells not only produce many molecules such as growth factors, cytokines and chemokines, but also play an important role through direct cellular interactions . In addition, proper differentiation and composition of different TECs are known to be critical for both thymocyte development and T-cell repertoire selection (Y. Takahama, Nat. Rev. Immunol. (2006), 6, 127-135).

The epidermal growth factor (EGF) -like domain is an evolutionarily well-preserved protein domain that has been implicated in blood coagulation, fibrinolysis, cell adhesion, And neural and vertebrate development [AK < RTI ID = 0.0 > Downing et al., Cell (1996), 85, 597-605]. For example, EGF-like repeats located in the extracellular domains of Notch receptors play an important role in regulating the Notch signaling pathway [S. Kojika et al., Exp. Hematol. (2001), 29, 1041-1052). These repetitive regions are associated with symptomatic ischemic cerebrovascular disease caused by mutations and polymorphisms in the EGF-like domain of the Notch3 protein And is known to be associated with the development of a number of serious illnesses [D. Ito et al., J. Neurol. Neurosurg. Psychiatry (2002), 72, 382-384].

EGFL7, a secretory protein expressed by endothelial cells as well as subset of neurons, plays a role in regulating angiogenesis during angiogenesis [D . Nichol et al., Blood (2010), 116, 6133-6143), proliferation of neural stem cells and self-renewal (M. Schmidt et al., Nat. Cell. Biol. (2009), 11, 873-880]. However, there is little known about the functional role of EGFL8, as opposed to some studies of EGFL7 function. Only that EGFL8 shows a high expression level in various tissues including the thymus with a similar expression pattern as EGFL7 as a potent homologous (paralog) of EGFL7 [M.J. Fitch et al., Dev. Dyn. (2004), 230, 316-324], recent studies have shown that EGFL8 expression is significantly reduced in colorectal and gastric cancer tissues, suggesting the use of EGFL8 as a novel biomarker for these cancers Is known [F. Wu et al., Anticancer Res. (2011), 31, 2249-2254; And F. Wu et al., Anticancer Res. (2011), 31, 3377-3380).

Thus, the present inventors have found that by in vitro testing using gain-of-function and loss-of-function techniques and in vivo testing using recombinant EGFL8 protein, Exemplary studies of the activity of EGFL8 in thymocyte and thymocyte epithelial cells (TECs) have shown that effective inhibition of T-cell mediated immunity in increasing or inhibiting the expression or activity of EGFL8 Or accelerating action, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a method for immuno-suppressing pharmacology capable of exhibiting immunosuppression or enhancing effect through effective inhibition or promotion action on T-cell mediated immunity by controlling the expression or activity of EGFL8 To provide a composition.

Another object of the present invention is to provide a method of immunomodulation using the composition.

It is another object of the present invention to provide a method for producing a recombinant EGFL8 protein.

In order to achieve the above object, the present invention provides a pharmaceutical composition for immunomodulation comprising an epidermal growth factor-like protein 8 (EGFL8) gene or an agonist or antagonist for a protein as an active ingredient.

In order to achieve the above other objects, the present invention provides an immunological composition comprising the above pharmaceutical composition, which is administered to a subject in need of immunomodulation to reduce the expression of the EGFL8 gene or protein in the subject or the activity of the EGFL8 protein Provides a method of adjustment.

According to another aspect of the present invention,

1) cloning the EGFL8 gene into an expression vector;

2) transforming the strain with the cloned expression vector;

3) culturing the transformed strain to express a recombinant EGFL8 protein; And

4) separating and purifying the expressed EGFL8 protein from the strain cell, and a method for producing the recombinant EGFL8 protein.

The present invention provides an immunomodulating pharmaceutical composition comprising agonists or antagonists of EGFL8 as an active ingredient and an immunomodulating method using the same, By modulating EGFL8 protein or EGFL8 protein expression by inhibiting the expression of EGFL8 gene or protein as a negative regulator important for development or by inhibiting the expression of Hes1 and Hey1 related to Notch signaling in the cell, Cell mediated immunity (T-cell mediated immunity), and thus can be used for the treatment and prophylaxis of inflammatory diseases such as cancer, rheumatoid arthritis and other autoimmune diseases, diabetes, dermatitis, graft-related diseases, As well as the prevention and treatment of a variety of diseases such as immune-related diseases such as depression, have.

FIG. 1 is a graph showing the results of transfection of EGFL8 expression vector into thymic epithelial cells (TECs) according to an embodiment of the present invention to overexpress EGFL8 to reduce intercellular adhesion molecule-1 (ICAM-1) As a result of confirming the inhibition of thymus epithelial cell-thymocyte adhesion, A is a culture medium (medium) and cell lysis (transfection) of HEK293 cells transfected with a vector Mock or pcDNA3.1-EGFL8-c-myc EGFL8 expression vector Western blot results performed using anti-c-myc antibodies against lysates; B was subjected to RT-PCR analysis of the EGFL8 gene in thymic subcapsular cortex or thymic nasal epithelial cells (SNECs) transfected with EGFL8 expression vector (Mock) or EGFL8 expression vector Result; C and D were obtained by co-culturing SNECs transfected with EMBL8 expression vector (Mock) or EGFL8 expression vector with thymocytes, and analyzed for thymocytes attached to SNECs. The number of thymocytes attached to the SNECs was counted by counting the number of thymocytes attached to SNECs. The number of thymocytes attached to the SNECs was counted. ; E is the result of RT-PCR analysis of the ICAM-1 gene in SNECs transfected with an empty vector (Mock) or EGFL8 expression vector (EGFL8); F is the result of FACS analysis of ICAM-1 expression on cell surface in SNECs transfected with EGFL8 expression vector (EGFL8).
FIG. 2 is a graph showing the effect of increasing EGFL8 expression by transfection of EGFL8 siRNA into thymus epithelial cells according to an embodiment of the present invention to increase thymic epithelial cell-thymocyte adhesion by increasing ICAM-1 expression , A is the result of RT-PCR analysis of the EGFL8 gene in SNECs transfected with scrambled siRNA (Scramble) or EGFL8 siRNA (siRNA); B and C are results of quantitative analysis of thymocytes attached to SNECs by co-culturing SNECs transfected with scrambled siRNA (Scramble) or EGFL8 siRNA (siRNA) with thymocytes, The arrows indicate the number of thymocytes attached to SNECs. C indicates the number of thymocytes attached to SNECs by cell counting method. D is the result of RT-PCR analysis of the ICAM-1 gene in SNECs transfected with scrambled siRNA (Scramble) or EGFL8 siRNA (siRNA); E is the result of FACS analysis of ICAM-1 expression on cell surface in SNECs transfected with scrambled siRNA (Scramble) or EGFL8 siRNA (siRNA).
Figure 3 shows that EGFL8 regulates T-cell development under in vitro conditions; A is a mock, EGFL8 expression vector (EGFL8), scrambled siRNA (Scramble) or EGFL8 siRNA ) Transfected SNECs were co-cultured with thymus cells and after 1 day (Day 1) or 2 days (Day 2), subtypes of thymocytes were treated with anti-CD4 and anti-CD8 antibodies The results were confirmed using the FACS analysis; B is confirmed the FACS analysis CD4 ^- CD8 ^- double negative (double-negative, DN), CD4 + CD8 + double-positive (double-positive, DP), CD4 + single positive (single-positive, SP) and CD8 ⁺ A graph showing the composition ratio of single positive (SP) thymocytes; C was analyzed by RT-PCR analysis of thymopoietic genes in SNECs transfected with EGFL8, EGFL8, Scramble or EGFL8 siRNAs (Mock, EGFL8, EGFL8 siRNA) .
FIG. 4 is a result of performing Coomassie blue staining (A) and Western blot analysis (B) by loading the recombinant mouse EGFL8 protein prepared according to an embodiment of the present invention on 10% SDS-PAGE.
FIG. 5 is a graph showing the results of a MALDI-TOF-MS / MS analysis of a recombinant mouse EGFL8 protein prepared according to an embodiment of the present invention by loading the protein on a 10% SDS-PAGE, cutting the band, Results.
Figure 6 shows the results of a test group of rat prostatic endothelial cells (rECs) (Cont) and recombinant mouse EGFL8 protein treated with 50, 150, 300, or 500 ng / ml of control group without treatment of recombinant mouse EGFL8 protein (0 hr) or 24 hr (24 hr) after treatment with EGFL8.
FIG. 7 shows the results of confirming the weight (A) of thymus tissue and the number of total thymocytes (B) by iv injection of 100 μg of recombinant mouse EGFL8 protein into mice.
Figure 8 shows thymocytes isolated from test group mice (EGFL-8) on days 1, 2 and 3 (Day 1, Day 2 and Day 3) after administration of non-administered control group (WT) and recombinant mouse EGFL8 CD4 and CD8 ^- monoclonal antibodies (mAbs) were treated with flow cytometry (A), and based on these results, four major subtypes of thymocytes, CD4 ^- CD8 ^- dual negative Quantitative analysis of absolute cell numbers of double-negative, DN, CD4 ⁺ CD8 ⁺ double-positive, CD4 ⁺ single-positive, SP and CD8 ⁺ to be.
FIG. 9 shows thymocytes isolated from test group mice after 12 hr (12 h), 24 hr (Day 1) and 72 hr (Day 3) administration of a non-administered control group (WT) and recombinant mouse EGFL8, (A) and a quantitative analysis (B) of the flow cytometry analysis, which confirmed the degree of cell proliferation by total thymocyte and thymocyte subtypes (DN, DP, CD4 SP and CD8 SP) )to be.
FIG. 10 shows thymocytes isolated from test group mice after 12 hr (12 h), 24 hr (Day 1) and 72 hr (Day 3) administration of the non-administered control group (WT) and recombinant mouse EGFL8. Annexin- Flow cytometry analysis using V-staining showed flow cytometry results (A) and quantitative analysis of apoptosis degree of total thymocyte and thymocyte subpopulations (DN, DP, CD4 SP and CD8 SP) B).
FIG. 11 shows the results of transmission electron microscopy (SEM) of the thymus isolated from the test group mice after 12 hours (B), 1 day (C) and 3 days (D) of the untreated control group (A) and the recombinant mouse EGFL8 electron microscope, TEM). The results are shown in Fig.
Figure 12 shows Western blot analysis of the expression of Hes1 (A) and Hey1 (B) after 12 hours of treatment of mouse thymic epithelial cells (TECs) treated with 100 ng / ml recombinant mouse EGFL8 protein .
FIG. 13 shows the results of the experiment after 12 hours (12 hrs), 1 day (Day 1) and 3 days (Day 3) of injection from the test group mice injected with untreated control mice and recombinant mouse EGFL8 protein (100 ng / Western blot analysis of the expression of Hes1 (A) and Hey1 (B) was performed on the thymus cells.

Hereinafter, the present invention will be described in detail.

The term "regulation" as used herein encompasses both positive or up regulation to promote any response and negative or down regulation that inhibits any response , Which means that any level of response can be improved or decreased through this modulation.

As used herein, the term "agonists " refers to substances, genes, proteins, compounds, etc. that act to increase the expression of a target gene or protein or the activity of a target protein, antagonists "refer to substances, genes, proteins, compounds, etc. that act to inhibit the expression of a target gene or protein or the activity of a target protein.

According to one embodiment of the present invention, it was confirmed that overexpression of EGFL8 in mouse thymocyte epithelial cells inhibits ICAM-1 expression and inhibits adhesion activity with thymocytes. In addition, this negative regulatory function of EGFL8 is not limited to cell adhesion between thymic epithelial-thymocyte cells, but also to the mature CD4 ⁺ and CD8 ⁺ single cells in an in vitro experimental model of mouse T- And the ratio of single positive (SP) thymocytes was also decreased. According to one embodiment of the present invention, when the EGFL8 expression in the thymus epithelial cells is knocked down through the RNA interference test, the number of thymocytes attached to the thymus epithelial cells and the number of thymocytes attached to the mature CD4 ⁺ and CD8 ⁺ Of the cells in the thymus epithelium, confirming the effect of EGFL8 modulation in the thymus epithelial cells.

Thymopoiesis is a key process for the development and maintenance of a healthy immune system because it is closely related to T-cell production and is regulated by a variety of factors including cytokines, hormones and chemokines Known [TS Heng et al., Curr. Opin. Pharmacol. (2010), 10, 425-433]. For example, IL-7 is a key thymocyte developmental cytokine that is predominantly expressed by MHC class II-expressing thymic epithelial cells and plays an important role in the development and homeostasis of T-cells Known [MA Oosterwegel et al., Immunity (1997), 6, 351-360; And K.S. Schluns et al., Nat. Immunol. (2000), 1, 426-432; GM-CSF is known to promote the development of T-cells through increased IL-6 expression associated with thymocyte activation [M. Papiernik et al., Eur. Cytokine Network (1992), 3, 89-95; TECK (CCL25) is a CC chemokine present in the thymic cortical epithelial cells and dendritic cells. It activates macrophages, Dendritic cells and thymocytes that cause T-cell development. Is known to exhibit chemotactic activity in cells [AP Vicari et al., Immunity (1997), 7, 291-301].

According to one embodiment of the present invention, overexpression of EGFL8 in an in vitro model of mouse T-cell development inhibits T-cell development through inhibition of ICAM-1 expression inhibition and expression of genes involved in thymocyte development . In addition, according to one embodiment of the present invention, it was confirmed that IL-7, GM-CSF and TECK expression levels in thymic epithelial cells can be increased through EGFL8 expression knockdown, whereby EGFL8 is expressed in thymus epithelial cells Lt; RTI ID = 0.0 > a < / RTI > negative regulator.

Also, according to one embodiment of the present invention, recombinant mouse EGFL8 protein is prepared and tested in vitro for rat prostatic endothelial cells (rECs), thymic epithelial cell line (TECs) and Biological activity was confirmed by in vivo tests on mouse thymus. As a result, The recombinant mouse EGFL8 protein showed a dose-dependent inhibitory effect on the migration of rECs (rat prostatic endothelial cells) as opposed to the paralogue EGFL7, It was confirmed that the number of thymocytes was significantly reduced. According to one embodiment of the present invention, the number of such thymocytes is determined by the number of CD4 ^- CD8 ^- double-negative (DN), CD4 ⁺ CD8 ⁺ double-positive, DP ), CD4 ⁺ single-positive (SP), and CD8 ⁺ monospecific (SP) cells were significantly reduced in the thymus when administered to mice, and this decrease was due to the cell proliferation of thymocytes of the recombinant EGFL8 cell proliferation inhibition and apoptosis induction.

According to one embodiment of the present invention, when the prepared recombinant mouse EGFL8 protein is treated or administered to mouse TECs, expression of Hes1 and Hey1 involved in Notch signaling pathway in mouse TECs and mouse thymocytes Of the total amount of water.

Thus, in vitro studies have shown that EGFL8 acts as a negative regulator in T-cell development through inhibition of ICAM-1 expression in TECs and inhibition of the expression of genes involved in thymocyte development And it was confirmed that endothelial cell migration could be inhibited when the recombinant EGFL8 protein was treated. In addition, in vivo experiments in which recombinant EGFL8 is administered to mice, EGFL8 strongly induces a decrease in thymus weight and thymocyte count, and the effect of this reduction in thymocyte counts on inhibition of thymocyte proliferation and It is confirmed that the cell death is induced by induction of apoptosis. In addition, it was confirmed that administration of recombinant EGFL8 inhibited the expression of Hes1 and Hey1, which are involved in the Notch signaling pathway in thymocytes and TECs.

Thus, when these results indicate that the expression or activity of EGFL8 gene or protein is regulated, the level of expression of various thymocyte developmental factors as well as ICAM-1 can be regulated, thereby inhibiting or promoting T-cell development . Accordingly, the immunomodulatory pharmaceutical composition comprising the EGFL8 gene or the protein agonist or antagonist for the protein according to the present invention can be used for the prophylactic treatment of the thymus through the modulation of ICAM-1 expression by increasing or decreasing the expression of EGFL8 gene or protein or the activity of EGFL8 protein. In addition to controlling cell interactions between cells and thymic epithelial cells, the development of mature CD4 ⁺ or CD8 ⁺ monocytic thymocytes is regulated through the regulation of expression of thymocyte developmental promoting genes such as IL-7, GM-CSF and TECK Cell mediated immunity. Therefore, the present invention provides a method for treating or preventing inflammatory diseases such as cancer, autoimmune diseases such as rheumatoid arthritis, diabetes, dermatitis, graft, Related diseases, and immune diseases such as immune deficiency diseases, as well as therapeutic agents for these fields Wide it may be utilized or the like.

Examples of EGFL8 gene or protein agonist or antagonistic substance contained in the pharmaceutical composition for immunomodulation of the present invention include genes capable of regulating the expression of EGFL8 gene or protein or controlling the activity of EGFL8 protein when applied in a cell, Protein, compound, or the like can be used.

Specifically, substances capable of regulating expression of the EGFL8 gene or protein include short interfering RNA (siRNA), which can increase or inhibit the expression of the EGFL8 gene or protein, including all or part of the sequence of the EGFL8 gene, DNA or RNA including cDNA, antisense RNA or DNA (antisense RNA or DNA; AS-RNA or AS-DNA), ribozyme, aptamer, plasmid DNA thereof or expression vectors thereof, Here, siRNA, AS-RNA or AS-DNA, expression vectors thereof and the like are used for suppressing the expression of EGFL8 gene or protein. EGFL8 cDNA, expression vector and protein thereof increase the expression of EGFL8 gene or protein . Examples of the substance capable of regulating the activity of the EGFL8 protein include a recombinant protein capable of enhancing or inhibiting the activity of the EGFL8 protein through direct or indirect interaction with the EGFL8 protein, , Peptides, polypeptides, vaccines, hormones, enzymes, ribozymes, cytokines or chemokines, compounds, drugs (including, for example, agents, and the like, but are not limited thereto. In addition, the activity of the EGFL8 protein may include an inhibitory activity against the expression of Hes1 and Hey1 involved in the Notch signaling pathway.

According to a preferred embodiment of the present invention, the agonist of EGFL8 used in the composition of the present invention is an expression vector containing all or a part of the sequence of EGFL8 gene or the antagonist of EGFL8 contains all or a part of sequence of EGFL8 gene ≪ / RTI >

In addition, according to a preferred embodiment of the present invention, the agonist of EGFL8 used in the composition of the present invention may be a recombinant protein comprising a part or whole sequence of EGFL8 protein. Therefore, the present invention provides a method for producing recombinant EGFL8 protein .

According to one embodiment of the present invention, a method for producing a recombinant EGFL8 protein comprises

1) cloning the EGFL8 gene into an expression vector;

2) transforming the strain with the cloned expression vector;

3) culturing the transformed strain to express a recombinant EGFL8 protein; And

4) separating and purifying the expressed EGFL8 protein from the strain cell.

According to an embodiment of the present invention, the expression vector may be a conventional expression vector such as pET28a, and the culture may be a conventional strain such as Escherichia coli including BL21 (DE3). In addition, since the recombinant EGFL8 protein is not only a protein secreted out of the strain cell but also a protein present in the cell precipitate, the protein isolated from the strain culture medium, as well as the expressed bacterial strain, is treated with a cell lysate and centrifuged All of the proteins may correspond to the recombinant EGFL8 protein.

In addition, according to one embodiment of the present invention, the recombinant mouse EGFL8 protein can have a Gly (2) -His (6) C-terminal label instead of the original signal peptide for purification, Ni-NTA column or the like can be used to perform protein purification.

According to one embodiment of the present invention, an immunomodulatory pharmaceutical composition comprising an agonist or antagonist of EGFL8 of the present invention as an active ingredient is administered to a mammal, including a human, Kg body weight, preferably 0.1 to 1 mg / kg body weight, in a single dose or in a number of divided doses per day through various forms of parenteral administration, including, but not limited to, And the dose or method may be suitably adjusted or selected depending on age, sex, weight, symptom, degree of disease, drug form, and duration of administration. Also, in some cases, dosage values less than the above-mentioned ranges may be more suitable and more doses may be used without causing harmful side effects.

The pharmaceutical composition of the present invention can be formulated in any form suitable for pharmaceutical preparations according to a conventional method, for example, in the form of sterile injection solution for intramuscular, intravenous, intraperitoneal or subcutaneous injection, And the like, or suppositories, etc., but the present invention is not limited thereto.

In addition, the therapeutic composition comprising the gene or the protein agonist or antagonist of EGFL8 of the present invention may further comprise appropriate carriers, excipients and diluents conventionally used for the formulation of the pharmaceutical composition depending on the administration form thereof When the therapeutic composition of the present invention is prepared in the form of an injection, examples of the carrier include water, saline solution, aqueous glucose solution, pseudosaccharide solution, alcohol, glycol, ether (e.g., polyethylene glycol 200) , Fatty acid esters, glycerides, surfactants, suspending agents, and emulsifiers.

The pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other pharmacologically active drugs or in any suitable combination.

The present invention also provides an immunomodulatory method comprising administering the pharmaceutical composition to a subject in need of immunomodulation, thereby reducing or increasing the expression of the EGFL8 gene or protein in the subject or the activity of the EGFL8 protein.

The pharmaceutical composition containing the EGFL8 gene or protein of the present invention as an active ingredient or an antagonistic substance as an active ingredient is administered to a subject including a mammal such as an experimental animal or a human such as immunity control such as immunosuppression or enhancement , The level of T-cell mediated immune response can be increased or suppressed by reducing or increasing the expression of EGFL8 in the subject in whole or in part. Examples of the subject in need of immunomodulation include cancer (liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, pancreatic cancer, thyroid cancer, cervical cancer, bladder cancer, testicular cancer, ), Rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, diabetes, atopic dermatitis, alopecia areata, psoriasis, pemphigus, asthma, aphtha stomatitis, chronic thyroiditis, aplastic anemia, cirrhosis, ulcerative colitis, The present invention provides a method of treating a patient suffering from an autoimmune hemolytic anemia, autoimmune hemolytic anemia, autoimmune encephalomyelitis, myasthenia gravis, hyperthyroidism, nodular polyarteritis, ankylosing spondylitis, atherosclerosis, , Graft-related diseases such as fibrotic diseases, temporal arteritis, asthma, graft-vesus-host disease (GVHD), and immune disorders such as immunodeficiency The number of experimental animals or patients that can exhibit or exhibit various inflammatory diseases such as arteriosclerosis, inflammatory bowel disease, obesity, osteoporosis, Alzheimer's disease, tuberculosis, sarcoidosis, hepatitis, cholecystitis, fungal infection, gastric ulcer, have.

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.

I. EGFL8 Overexpression or suppression of the expression of the immunomodulatory effect: In vitro in  vitro ) exam

Materials and methods

1) Cell and cell culture

Generation, maintenance and functional characterization of the thymic subcapsular cortex or thymic nurse epithelial cells 427.1 (SNECs) were performed according to the method of Faas et al. [SJ Faas et al., Eur. J. Immunol. (1993), 23, 1201-1214]. The cell line expresses class I antigens of SV40 T antigen transgene and major histocompatibility complex (MHC) structurally and stimulates the interaction of recombinant interferon (IFN) -γ. Can be induced to express MHC class II antigens and produce granulocyte-macrophage colony-stimulating factor (GM-CSF) [SJ Faas et al., Eur. J. Immunol. (1993), 23, 1201-1214]. These cell lines were developed by Dr. Barna B. Knowles (The Jackson Laboratory, Bar Harbor, ME, USA). All cell lines used in the following examples were inoculated with 10% v / v fetal bovine serum (Gibco, Invitrogen), 2 mM glutamine (Sigma, St. Louis, Mo., USA), 100 U / ml penicillin (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 100 ug / ml streptomycin (Gibco, Invitrogen) high glucose DMEM (high glucose Dulbecco's modified Eagle ◎ medium) , And 5% CO _{2 at} atmospheric conditions.

2) Plasmid preparation and transfection

To construct the EGFL8 plasmid, the full-length mouse EGFL8 cDNA was amplified with total thymus RNA and inserted into the pcDNA3.1 mammalian expression vector for cloning. The cloned pcDNA3.1-EGFL8 or pcDNA3.1-EGFL8-c-myc expression construct was transfected into the cell line using lipofectamine 2000 (Lipofectamine 2000, Invitrogen) according to the manufacturer's instructions. After 48 hours of culture, cells were selected for 14 days with 750 / / ml of G418 sulfate (G418 sulfate, Invitrogen) as needed.

3) siRNA transfection

For knockdown of the EGFL8 gene, negative control siRNAs with 21-bp siRNA duplexes and dTdT overhangs were prepared from Bioneer (Daejeon, Korea), and the prepared EGFL8 siRNA The sequence is the same as the sequence of SEQ ID NO: 1. SNECs cells were seeded in 6-well microplates and transfected with 100 pmol of siRNA according to the manufacturer's instructions using lipofectamine 2000.

4) Semiquantitative reverse transcription-polymerase chain reaction analysis

Total RNA was isolated from cultured cells according to the manufacturer's instructions using trizol reagent (Ambion, Invitrogen). (Moloney murine leukemia virus) reverse transcriptase (Promega) and oligo (dT) [oligo (dT)] primers to perform reverse transcription-polymerase chain reaction (RT-PCR) Single-strand cDNA was synthesized from 2 ㎍ of total RNA. All PCR reactions included EGFL8 forward and reverse primers of SEQ ID NOs: 2 and 3, ICAM-1 forward and reverse primers of SEQ ID NOs: 4 and 5, IL-7 forward direction of SEQ ID NOs: 6 and 7, Reverse primers of SEQ ID NOs: 8 and 9, TECK forward and reverse primers of SEQ ID NOs: 10 and 11, or GAPDH forward and reverse primers of SEQ ID NOs: 12 and 13 at 94 DEG C 30 seconds at 55 占 폚 for 30 seconds, and 72 占 폚 for 30 seconds was performed in 22 to 35 cycles. After PCR, reaction product samples were analyzed by agarose gel electrophoresis.

5) Western blot analysis

Total protein was extracted from cultured cells using protein extraction solution (Intron, Seoul, Korea) containing protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA) The concentration of the extracted protein was measured using a Bradford protein assay kit (Bio-Rad, Hercules, Calif., USA), and the same amount of protein samples were analyzed with SDS (sodium dodecyl sulfate) polyacrylamide gels and electroblotted onto a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA). The blotted membrane was blocked with 0.1% Tween 20 in Tris-buffered saline (0.1% Tween 20, TBS-T) containing 5% skim milk, The reaction was carried out overnight at 4 ° C with a secondary antibody and reacted with HRP-labeled secondary antibody (horseradish peroxidase-conjugated) for 1 hour at room temperature. The immunoreactivity of the reaction membrane was confirmed by an enhanced chemiluminescent substrate kit (Pierce, Rockford, Ill., USA), and the analyzed image was captured and analyzed using a LAS-3000 imaging system (Fujifilm, Tokyo , Japan).

6) Quantification of thymic epithelial cell adhesion (TECs)

Adhesion of thymocytes to TECs was assessed by the conventional method [M. Barda-Saad et al., Exp. Hematol. (1996), 24, 386-391; And HW Lee et al., Regul. Pept. (2008), 147, 72-81]. Freshly isolated thymocytes from C57BL / 6 mice were seeded at 1x10 ⁷ cells / well in DMEM containing 10% FBS on SNECs cell line layer in 6-well microplates and plates were incubated for 4 hours. Thymocytes not attached to SNECs were carefully washed and washed with PBS (phosphate-buffered saline), and thymocytes attached to SNECs were recovered by treatment with PBS containing 0.5 mM EDTA (Sigma) for 2-3 minutes. The number of thymocytes was measured via trypan blue staining followed by a hemocytometer. For in vitro T-cell development studies, thymocytes in SNECs cell line were further cultured for 1-2 days and recovered for FACS (flow cytometry) analysis.

7) FACS (flow cytometric analysis) analysis

Cells were pre-incubated with anti-FcγRII / III (2.4G2, hybridoma supernatant) to avoid nonspecific binding to antibodies and then incubated in HBSS containing 2% FBS (Gibco, Invitrogen) Lt; / RTI > for 30 minutes with ice-cooling. Background fluorescence was determined through cells that were reacted with isotype-matched nonreactive antibodies. FACS analysis of antibody-reactive cells was performed using FACSCanto II (BD Biosciences, San Jose, CA, USA) and the results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

8) Statistical analysis

All results were expressed as mean ± SD (SD) for each analysis. For the results of comparative tests in several groups, Scheffe's post hoc test was performed after one-way analysis of variance (ANOVA). Statistical significance was evaluated based on p <0.05.

Test Example  One: EGFL8  Over-expression Thymocyte - Thymic epithelial cells  Inhibition of adhesion

Since the EGFL8 protein is known to be a secreted protein through structural analysis, M.J. Fitch et al., Dev. Dyn. (2004), 230, 316-324], and EGFL8 cDNA synthesized from mouse thymus tissue was cloned to construct pcDNA3.1-EGFL8-c-myc EGFL8 expression vector. Transient transfection of HEK293 cells transfection). One day after the transfection, cell culture medium and cell lysate of HEK293 cells injected with EGFL8- were analyzed by Western blot using anti-c-myc antibody, respectively. As a result, as can be seen from FIG. 1A, in the case of HEK293 cells transiently transfected with pcDNA3.1-EGFL8-c-myc expression vector, EGFL8 protein could be identified in both cell culture medium and cell lysate, As a result, the EGFL8 protein was found to be a secreted protein.

In order to confirm the activity of EGFL8 in mouse thymus epithelial cells, SNECs were transfected with pcDNA3.1-EGFL8 or pcDNA3.1 empty vector, and stable cell lines were established by G418 selection , And the efficacy of EGFL8 expression in SNECs was confirmed by RT-PCR analysis (Fig. 1B).

As shown in FIGS. 1C and 1D, the effect of EGFL8 on the ability of thymocytes to adhere to SNECs cells was assessed through cell adhesion assay using established cell lines. As a result, , The number of thymocytes attached to the thymus epithelial cells was significantly reduced in the case of EGFL8-overexpressing SNECs.

In addition, EGFL8 is known as one of the key molecules involved in thymocyte-thymus epithelial cell adhesion [S. Marlin et al., Cell (1987), 51, 813-819], the expression level of ICAM-1 mRNA in EGFL8-transfected SNECs was analyzed by RT-PCR As a result, it was confirmed that EGFL8 overexpression inhibited the expression of ICAM-1 in SNECs as shown in FIG. 1E, and the ICAM-1 level on the surface of SNECs was also reduced by FACS analysis F).

Thus, it can be seen that EGFL8 inhibits thymocyte adhesion to thymic epithelial cells by decreasing ICAM-1 expression level.

Test Example  2: EGFL8  Knockdown knockdown )through Thymocyte - Thymic epithelial cells  Acceleration of attachment

To further confirm the activity of EGFL8 associated with thymocyte adhesion to thymic epithelial cells, EGFL8-specific siRNA (EGFL8 siRNA) was used to knockdown expression of EGFL8 in SNECs, resulting in adhesion between thymocytes and thymic epithelial cells The results are as follows. First, the RT-PCR analysis of siRNA efficacy against EGFL8 expression inhibition showed that control scrambled siRNA treatment had no effect on EGFL8 expression (FIG. 2 A), whereas EGFL8 siRNA inhibited expression of EGFL8 in SNECs Lt; RTI ID = 0.0 &gt; expression &lt; / RTI &gt; Thus, specific inhibition of expression through RNA interference of EGFL8 was observed, confirming the activity of EGFL8 associated with thymocyte adhesion to SNECs.

After the transfection was performed using the confirmed EGFL8 siRNA, the EGFL8 effect on the thymocyte adhesion ability of SNECs was examined through cell adhesion test. As a result, as shown in FIGS. 2B and C, the EGFL8 When the expression was knocked down, thymocyte adhesion to thymic epithelial cells was significantly increased. In addition, in order to examine whether siRNAs also affect ICAM-1 expression in SNECs, ICAM-1 mRNA and protein expression levels were analyzed by RT-PCR and FACS analysis, respectively. As a result, Similarly, expression of ICAM-1 was significantly increased in EGFL8 siRNA-transduced SNECs.

Test Example  3: Thymic epithelial cells  of mine EGFL8  In vitro by expression ( in vitro ) Inhibition of T-cell development

Based on the above EGFL8 inhibition of thymocyte adhesion to mouse thymus epithelial cells through the decrease of ICAM-1 expression, we confirmed whether EGFL8 regulates the development of T-cells in vitro. Thymus cells freshly isolated from mice were co-cultured by dividing them into monolayers of EGFL8-overexpressing or EGFL8 siRNA-transfected SNECs, and then floating thymocytes were recovered to obtain mouse CD4 and CD8 Lt; RTI ID = 0.0 &gt; monoclonal &lt; / RTI &gt; As a result, as can be seen in FIGS. 3A and 3B, in the case of thymocytes co-cultured with EGFL8-overexpressing SNECs, CD4 ⁺ and CD8 ⁺ monoclonal (compared to the case of empty vector-transfected control SNECs) single-positive, SP) thymocyte was decreased, and in the case of thymocytes co-cultured with siRNA-transfected SNECs, the proportion of CD4 ⁺ and CD8 ⁺ SP individuals was scrambled with siRNA-transfected SNECs compared with control group.

Based on these results, EGFL8-overexpressed and EGFL8-silenced SNECs cell lines were screened for IL-7, GM-CSF and EGFL8 in order to confirm whether EGFL8 can regulate the expression of thymopoietic factors TECK expression was analyzed by semi-quantitative RT-PCR analysis. As a result, as shown in FIG. 3C, the expression of IL-7, GM-CSF and TECK in SNECs was significantly increased by EGFL8 knockdown, whereas expression of these factors was suppressed by EGFL8 overexpression Respectively.

Ⅱ. Recombinant mouse EGFL8  Production and identification of proteins

Materials and methods

1) Reagent

Most enzymes and reagents for cloning were purchased from Life Technologies (Grand Island, NY, USA) and New England Biolabs (Ipswich, MA, USA). Escherichia coli BL-21 (DE3) and pET28a vectors were obtained from Novagen / VWR International (Radnor, PA, USA) and in RNA purification kit, Ni-NTA column and plasmid DNA isolation Plasmid DNA isolation kit was purchased from Qiagen (Valencia, CA, USA). (D-thiogalactopyranoside, IPTG, Roche, Meylan, France), Bradford protein assay kit (Bio-Rad, Hercules, CA, USA), PVDF membrane and Brilliant Blue R-staining solution (Elpis Biotech, Daejeon, South Korea) were purchased and used. The other remaining compounds were purchased from Sigma- Were purchased from Aldrich (St. Louis, MO, USA).

2) SDS-PAGE and Western blot

Protein concentrations were measured using a Bradford protein assay kit. Protein samples were then separated on SDS-PAGE and stained with Brilliant Blue R-staining solution, and then the bots were electrically transferred onto a PVDF membrane. The membrane was immersed in TBS-T (containing 0.1% Tween 20) containing 5% non-skimmed milk and blocked with polyclonal EGFL8 primary antibody (Sigma-Aldrich) at 4 ° C After overnight incubation, the cells were washed and reacted with secondary HRP-labeled anti-mouse IgG antibody (1: 10000) for 1 hour at room temperature. Immunoreactivity was confirmed using an ECL kit (Pierce, Rockford, Ill., USA), and images were developed and quantitatively analyzed on a LAS-3000 imaging system (Fujifilm, Tokyo, Japan).

3) Protein weight spectrometric analysis

EGFL8 protein was loaded onto 10% SDS-PAGE and stained with Coomassie blue. The protein bands were cut out and put into microcentrifuge tubes containing 0.5 ml of distilled water, The gel was dissolved by treatment and MALDI-TOF-MS / MS analysis was performed on the gel.

Manufacturing example  1: Recombinant mouse EGFL8  Protein Expression and Isolation Purification

1) Mouse EGFL8 gene cloning

To isolate the coding sequence of the mouse EGFL8 gene, total cDNA (0.8 kb) was isolated and PCR amplified and cloned into pcDNA3.1 (Life Technologies). The PCR amplification was to start (start) and finalization (stop) codon (codon) Nco1 and Xho1 restriction enzyme recognition sequence was created in a position using the SEQ ID NO: 2, and 3 of EGFL8 the forward and reverse primers, wherein the primers each 10 ng, 0.1 mM dNTP and 5 U Pfu The final reaction mixture containing DNA polymerase was denatured at 94 ° C for 30 seconds and then reacted at 94 ° C for 30 seconds; 60 캜, 30 seconds; The reaction was carried out at 72 ° C for 1 minute in 25 cycles. End 72 ℃, after gel analysis of the amplification product after completion of the polymerization reaction of two minutes Nco1 and Xho1 Restriction enzyme cleavage was performed. The resulting product was separated by agarose gel electrophoresis and purified with a QIAquick gel extraction kit to obtain a C-terminal His · Tag / Thrombin / T7 · Tag sequence and a selective C-terminal His · Tag sequence (C- terminal His · Tag / thrombin / T7 · Tag configuration plus an optional C-terminal His sequence. NovaBlue host cells (5x10 ⁶ cells) were transformed with the inserted vector and cultured on LB (Luria broth) agarose plates containing 70 μg / ml kanamycin. Plasmid DNA was then isolated from positive clones and analyzed by 1.5% agarose gel. Both positive clones were amplified to obtain cloned plasmid DNA (pET28a-EGFL8).

2) Expression and isolation of recombinant mouse EGFL8 protein

BL21 (DE3) cells were transformed with the cloned pET28a-EGFL8 and then cultured overnight at 37 ° C in 50 ml of LB medium containing 10 μg / ml kanamycin on a rotary table (220 rpm) And inoculated in one liter of fresh LB medium. After incubation for 2 hours, 0.2 mM IPTG was added thereto to promote the expression of EGFL8 protein and cultured for another 4 hours. Cultured E. coli cells were obtained by centrifugation at 6000 rpm for 8 to 10 minutes.

The obtained cell pellet was cryopreserved at -80 ° C. until purification. The pellet to be subjected to protein purification was dissolved in 60 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA) Lt; / RTI &gt; After adding 0.5% Triton X-100, 0.1 mM PMSF and 1 mM DTT (dithiothreitol), the mixture was pulsed with 50% Decomposition was performed and then centrifuged at 12000 rpm for 15 minutes. The supernatant was discarded, and the pellet was resuspended in the dissolution buffer, except that a solution containing 10 mM MgCl ₂ , 0.01 mg / ml DNase, and 0.1 mg / ml lysozyme was added to the mixture of Triton X-100, PMSF and DTT And the process was repeated. The pellet mixture was allowed to react at room temperature for 20 minutes, followed by sonication and centrifugation as above. The supernatant was removed and the pellet was resuspended in a 60 ml dissolution buffer. The pellet was resuspended in 40 ml of an 8 M urea solution (100 mM Tris-HCl, pH 8.0, 50 mM glycine), and the supernatant was removed by centrifugation. It was resuscitated.

The protein was completely dissolved by shaking it slowly at room temperature for 1 hour, and insoluble proteins were removed by short centrifugation. The concentration of the obtained protein was analyzed using Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). To restore the biological activity of the recombinant protein, a protein concentration of 1 to 2 mg / ml was diluted with 2 liters of dialysis buffer buffers: 20 mM Tris-HCl-pH 8.0, 150 mM NaCl and 0.1 mM DTT) at 4 [deg.] C for 12-15 hours. At this time, the same buffer except for DTT was used as the third dialysis buffer.

3) Purification of recombinant mouse EGFL8 protein through Ni-NTA column

The recombinant mouse EGFL8 protein had the Gly (2) -His (6) C-terminal label instead of the original signal peptide for purification, and was purified as follows.

The column was washed with distilled water and washed with 6 × Ni-NTA washing buffer (washing buffer: 50 mM NaH ₂ PO ₄ , pH 8.0; 300 mM NaCl; 20 mM imidazole] was loaded. The dialyzed protein mixture was slowly passed through the Ni-NTA column and bound EGFL8 protein was eluted with 6 x Ni-NTA elution buffer (50 mM NaH ₂ PO ₄ , pH 8.0, 300 mM NaCl ; 250 mM imidazole). The eluted proteins were identified by SDS-PAGE.

Test Example  4: Manufactured recombinant mice EGFL8  Protein identification

The recombinant mouse EGFL8 protein prepared above was loaded onto 10% SDS-PAGE and Coomassie blue staining and western blot analysis were performed.

As a result, as can be seen in FIG. 4A, a single protein band of 30.09 kDa was confirmed, indicating that the recombinant EGFL8 protein was successfully purified. In addition, as can be seen in Figure 4B, the protein was analyzed by Western blot to reaffirm the mouse EGFL8 protein.

In addition, the EGFL8 protein band loaded on 10% SDS-PAGE was excised and subjected to tryptic treatment, followed by mass spectrometry of the Malidophs.

As a result, it was confirmed that the weight of the purified EGFL8 protein was 30.09 kDa, which was the same as the theoretical value, as can be seen from the Maldiston mass spectrometry data (data) of FIG.

Ⅲ. Recombinant mouse EGFL8  In vitro and in vivo administration of proteins to confirm immunomodulatory effects: In vitro in vitro ) And in vivo ( in vivo ) exam

Materials and methods

1) Cell line and cell culture

Mouse thymic cortical reticular epithelial cells (1308.1; CREC) and mouse thymic medullary epithelial cells (6.1.1; MEC) were prepared according to the method known by Faas et al. (1993) These cells were cultured in DMEM. And were supplied by Barbara B. Knowles (The Jackson Laboratory, Bar Harbor, ME, USA). Rat prostatic endothelial cells (rECs, YPEN-1), also known by Yamazakai et al. (1996), were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All the cell lines were cultured in DMEM (GIBCO BRL, Grand BRL) containing 100% U / ml penicillin (GIBCO BRL) and 100 ug / ml streptomycin (GIBCO BRL) ) Medium at 37 ° C in a 5% CO ₂ incubator.

2) Cell migration assay (Cell migration assay)

rECs were cultured in 6-well culture plates. When rECs are cultured under standard conditions, confluence occurs and wounds are generated. Thereafter, the cells were washed with PBS, and the cells were treated with DMEM containing 2% FBS (FBS concentration in which cells survived but not proliferated), EGFL8 was not treated, treated at 50, 150, 300 or 500 ng / Lt; / RTI &gt; The cultured cells were each washed with PBS and observed on an inverted microscope and analyzed.

3) In vivo treatment of experimental animals and EGFL8

Adult male, specific pathogen-free, and C57BL / 6 mice were purchased from Dae Han Bio Link, Seoul, Korea. The mice were placed in a cage at a rate of 24 hours at a temperature of 24 ° C for 12 hours per person / cancer cycle, a specific pathogen member, and a water-regulating facility. Basically, sterilized food and water were randomly ad- libitum ). The mice were adapted to the given environment for one week and then used at the time of 8-10 weeks of age. Recombinant mEGFL-8 (100 [mu] g) was administered intravenously (iv) to the experimental animals with physiological saline and the control group was administered only saline. The experiment was carried out after 4 or more mice were sacrificed at 12 hours, 1, 2 and 3 days after administration of the injections. The control and experimental procedures for these experimental animals were carried out in accordance with "Animals Experimental Guidelines &quot;.

After weighing, the animals were anesthetized by intraperitoneal (ip) injection of sodium pentobarbital (5 mg / kg body weight) and sacrificed. The absolute weight of the thymus ) Were recorded.

4) Proliferation assay

Bromo-2'-deoxyuridine (BrdU, BD Biosciences-Pharmingen, San Jose, Calif., USA) USA) solution was intraperitoneally injected and 100 ~ 200 μl of physiological saline was injected into the control group. BrdU incorporation can be detected slowly in the thymus and bone marrow from 1 hour after injection. Two hours after BrdU injection, the thymus tissue was dissected by incision and thymocytes were passed through cell strains to obtain single-cell suspensions. After counting the number of cells obtained, 5 × 10 ⁵ cells were stained for CD4, CD8, CD25, CD44 and BrdU according to the manufacturer's manual using a BrdU flow kit (BD Biosciences-Pharmingen) And analysis of thymocyte subsets was performed.

5) Apoptosis assay

Annexin V-FITC staining was performed according to the manufacturer's protocol using anti-Annexin V Detection Kit (Cat. No. 51-65874X, BD Biosciences-Pharmingen) as follows.

5 μl of 5 × 10 ⁵ cell suspension was placed in a 5 ml tube, 5 μl of annexin V-FITC and 5 μl of propidium iodide were added thereto, gently vortexed, Lt; / RTI &gt; for 15 min. Then, 400 [mu] l of 1x binding buffer was added to each tube, and flow cytometry was performed within 1 hour.

6) Flow cytometric analysis

Fluorochrome-conjugated monoclonal antibodies (mAbs), i.e., Pacific blue-conjugated anti-CD4 antibodies (RM4-5), allophycocyanin-Cy7-linkage CD8 antibody (PE labeled anti-CD25, PC61) and APC anti-CD44 antibody (APC anti-CD44, IM7 ) Were purchased from BD Biosciences-Pharmingen.

Thymocytes and the fluorescence-conjugated antibodies were reacted for 30 min in a solution of HBSS (Hank's balanced salt solution, Life Technologies) containing 2% FBS on ice. Background fluorescence was detected by isotype-matched nonreactive antibodies ). &Lt; / RTI &gt; Flow cytometry was performed using FACSCanto II (BD Biosciences-Pharmingen), and the obtained results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

7) Electron Microscopy Analysis

For electron microscopic analysis, the experimental animals were anesthetized with an ip injection of pentobarbital sodium (5 mg / kg body weight). Thymus tissue was fixed at a rate of 5 ml / min in a peristaltic pump by vascular perfusion. The ascending aorta was inserted through a left ventricle into a 16-gauge polyethylene insertion tube (16- gauge polyethylene catheter). First, the blood vessels were washed with 0.1 M phosphate buffer (pH 7.4) and fixed with 0.1 M phosphate buffer (pH 7.4) containing 1.6% paraformaldehyde and 1.7% glutaraldehyde for 20 minutes The thymus tissue was then cut out, thinned with a razor blade, and fixed at 4 ° C for 2 hours in the same manner as described above. The tissue pieces were rinsed with 0.1 M phosphate buffer (pH 7.4) and fixed with 0.1 M phosphate buffer (pH 7.4) containing 1% osmium tetroxide at 4 ° C for 90 minutes. Fixed biopsy tissues were treated with upgraded alcohols and propylene oxide according to conventional methods and embedded in Epon 812 resin, which was then placed in an ultra-fine cutter equipped with a diamond knife ultramicrotome, LKB, NOVA, Bromma, Sweden) to obtain ultrafine slices. Sections showing gray to silver interference colors were placed on unsupported 200-mesh copper grids and then double-coated with uranyl acetate and lead citrate, And the sections were analyzed using a JEOL-1200 EXII transmission electron microscope.

8) Preparation of protein samples of thymocytes and thymus epithelial cells (TECs)

Thymic epithelial cells (TECs) were inoculated into 5 × 10 ⁵ cells in a 100-mm dish containing DMEM containing 10% FBS and treated with 100 ng / ml of EGFL8 for 12 hours. In addition, the mice injected with 100 ng / ml of EGFL8 were sacrificed 12 hours, 1 day, or 3 days later, and thymocytes were isolated and recovered. The obtained thymocytes or thymus epithelial cells (TECs) were washed with cold PBS, and then a protein extraction solution (Protein Inhibitor Mixture, Roche) was used (Intron, Seoul, Korea) The total protein was extracted, and the concentration of the extracted protein was confirmed using a Bradford protein assay kit (Bio-Rad).

9) Western blot analysis

Protein samples of the same volume were placed in a sample buffer using a Mini-Protean III system (Bio-Rad), heat-treated at 95 ° C for 10 min and then separated by 12% SDS-PAGE. The PVDF membrane (Immobilon-P, Millipore) (Wet transfer, Bio-Rad). (Anti-Hes-1, sc-166378, Santa Cruz Biotechnology, Santa Cruz, Calif.) After blocking the bluntted membrane with TBS-T containing 5% skim milk for 1 hour at room temperature. CA, USA), anti-Hey-1 antibody (anti-Hey-1, sc-28746, Santa Cruz Biotechnology) or anti-β actin antibody (Abcam, Cambridge, UK) The reaction was carried out overnight. The goat anti-mouse IgG-HRP conjugated antibody (goat anti-mouse IgG-HRP conjugate antibody, 7076, Cell Signaling Technology, Boston, Mass., USA), a secondary antibody, was washed with TBS-T 3 times And reacted at room temperature for 1 hour, and then re-washed three times with TBS-T. Immunoreactivity was confirmed using an ECL substrate kit (Pierce, Rockford, IL, USA), and images were captured and quantitatively analyzed using a LAS-3000 imaging system (LAS-3000 imaging system; Fujifilm, Tokyo, Japan) .

10) Statistical analysis

All results were expressed as mean ± SD (SD) for each analysis. Student t-test was performed on the results of comparative tests in several groups, and statistical significance was evaluated based on p <0.05.

Test Example  5: Recombination EGFL8  Identification of biological activity of protein

1) Cell migration assay (cell migration assay)

Since it is known that EGFL7, a paralogue of EGFL8, plays an important role in the activities such as proliferation and migration of endothelial cells, the migration of endothelial cells ).

As a result, as shown in FIG. 6, the rCEs treated with the recombinant mouse EGFL8 protein showed a dose-dependent inhibitory effect on cell migration.

2) Confirmation of effect of recombinant EGFL8 on thymus weight and number of thymocytes

To confirm the effect of recombinant EGFL8 protein on mouse thymocytes, mice were injected with 100 [mu] g EGFL8 protein i.v. And weights of thymus tissue and the number of total thymocytes were confirmed by injection.

As a result, as shown in FIG. 7, it was confirmed that the thymus tissue weight and the number of thymocytes were remarkably decreased after the injection of recombinant EGFL8, and this reduction effect started from the 1st day after injection of EGFL8 and reached the maximum value at the third day Respectively.

3) The effect of recombinant EGFL8 on the four major subtypes of mouse thymocytes

After confirming that the number of thymocytes was significantly decreased after the administration of recombinant EGFL8, changes in the four major subtypes of thymocytes were confirmed by treatment with recombinant EGFL8. For this purpose, mouse thymocytes were isolated on days 1, 2 and 3 after injection of EGFL8 into test mice, and analyzed for constitutive units by treatment with anti-CD4 and anti-CD8 mAbs.

As a result, as shown in FIGS. 8A and 8B, when mouse thymocytes were isolated from days 1, 2, and 3 after injection of EGFL8, anti-CD4 and anti-CD8 mAbs were treated with CD4 ⁺ CD8 ⁺ DP thymocytes were dramatically decreased, and the CD4 ⁺ SP and CD8 ⁺ SP thymocyte units were also much weaker than the CD4 ⁺ CD8 ⁺ DP units, but significantly decreased compared to the untreated control group.

4) Confirmation of effect of recombinant EGFL8 on proliferation or death of mouse thymocyte

After confirming that the number of thymocytes decreased significantly after administration of recombinant EGFL, it was confirmed whether these changes were due to inhibition of cell proliferation of thymus cells or promotion of cell death by treatment with recombinant EGFL8. To confirm the effect of recombinant EGFL8 on thymocyte proliferation, cell proliferation assay was performed at 12, 24 and 72 hours after EGFL8 administration. For this, BrdU injection was performed 2 hours before analysis, and thymocytes isolated from thymus were analyzed for staining for CD4, CD8, CD25, CD44 and BrdU.

As a result, as shown in FIG. 9, it was confirmed that the numbers of BrdU ⁺ thymocytes, which are markers of proliferating cells in all of the DN, DP, CD4 ⁺ SP and CD8 + SP thymocyte units after 12 and 24 hours of EGFL8 injection , Indicating that the recombinant EGFL8 exhibited remarkable cell proliferation inhibitory effects on all four constitutive units of thymocytes. However, it was confirmed that the number of BrdU ⁺ thymocytes recovered similarly to that in the control mice on the third day after administration of EGFL8.

In addition, to confirm whether recombinant EGFL8 induces apoptosis in thymocyte development, thymocytes isolated after 12 hours, 1 day and 3 days of recombinant EGFL8 injection were incubated with antibodies to annexin-V Analysis was performed by staining with anti-CD4 and anti-CD8 mAb.

As a result, as shown in FIG. 10, it was confirmed that apoptosis was significantly induced in all four subtypes of thymocytes, although it was relatively low in all the periods tested after the injection of recombinant EGFL8. After a period of time, it was confirmed that annexin-V ⁺ cells reached the maximum value. This indicated that recombinant EGFL8 acts as a strong apoptotic derivative for mouse thymocytes.

5) Ultrastructural alteration of the thymus by recombinant EGFL8

Transmission electron microscopy (TEM) was performed after injection of EGFL8 to determine whether the recombinant EGFL8 protein causes morphological changes at the ultrastructural level in mouse thymus.

As a result, as shown in FIGS. 11A to 11D, the thymus of the control mice showed a typical hyperfine structure under the TEM analysis, whereas the EGFL8 was administered 12 hours after the administration of the EGFL8, 11 (B), although these sites were relatively small, it was confirmed that these killed cells showed typical morphological changes in which chromatin condensed and aggregated into large, black compacts . On the other hand, most of the killing cells were found in thymocytes, while in the TECs and other thymic cell types, there were few killing cells. In addition, it was confirmed that the number of death thymocytes on the first day after EGFL8 administration was much smaller than that of 12 hours after administration (Fig. 11C), whereas the third day of recombinant EGFL8 administration, It was also confirmed that the microstructure exhibits a normal shape in general (Fig. 11D).

6) Confirmation of effect of recombinant EGFL8 on Hes1 and Hey1 expression in mouse TECs and thymocytes

To confirm the effect of recombinant EGFL8 on the Notch signaling pathway in mouse TECs, TECs were treated with recombinant EGFL8 protein at 100 ng / ml for 12 hours to Western blot analysis of Hes1 and Hey1 expression Respectively.

As a result, as shown in FIG. 12, it was confirmed that the expression of Hes1 and Hey1 in TECs was strongly inhibited by the treatment of EGFL8.

In order to examine the effect of recombinant EGFL8 on the Notch signaling pathway in mouse thymocytes, the expression of Hes1 and Hey1 in thymocytes isolated from mice injected with recombinant EGFL8 protein (100 ng / ml) Western blot analysis was performed.

As a result, as shown in FIG. 13, it was confirmed that the expression of Hes1 and Hey1 in mouse thymocytes was remarkably reduced due to the administration of recombinant EGFL8. The inhibitory effect of recombinant EGFL8 on Hes1 was observed 12 hours after injection (Fig. 13A). In the case of Hey1, the most remarkable effect was observed on the first day after injection (Fig. 13B).

Thus, in vitro studies have shown that EGFL8 acts as a negative regulator in T-cell development through inhibition of ICAM-1 expression in TECs and inhibition of the expression of genes involved in thymocyte development And it was confirmed that endothelial cell migration could be inhibited when the recombinant EGFL8 protein was treated. In addition, in vivo experiments in which recombinant EGFL8 is administered to mice, EGFL8 strongly induces a decrease in thymus weight and thymocyte count, and the effect of this reduction in thymocyte counts on inhibition of thymocyte proliferation and It is confirmed that the cell death is induced by induction of apoptosis. In addition, the administration of recombinant EGFL8 inhibited the expression of Hes1 and Hey1, which are involved in the Notch signaling pathway in thymocytes and TECs.

Thus, results of these examples show that the immunomodulatory pharmaceutical composition comprising the agonist or antagonist for EGFL8 according to the present invention increases or decreases the expression of the EGFL8 gene or protein or the activity of the EGFL8 protein, (Liver, stomach, colon, lung, breast, ovarian, prostate, kidney, and prostate), which require immune control such as immunosuppression or enhancement, Pancreatic cancer, thyroid cancer, cervical cancer, bladder cancer, testicular cancer, skin cancer, etc.), rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosis, diabetes mellitus, atopic dermatitis, alopecia areata, psoriasis, pemphigus, asthma, aphtha stomatitis, chronic thyroiditis, , Ulcerative colitis, Vacchet's disease, Crohn's disease, Silicoside, Asbestosis, Kidney disease, Glomerulonephritis, Sjogren's syndrome, - graft-versus-host disease, vesicular syndrome, dermatomyositis, multiple myositis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, myasthenia gravis, hyperthyroidism, nodular polyarteritis, ankylosing spondylitis, inflammatory bowel disease, obesity, osteoporosis, Alzheimer's disease, tuberculosis, sarcoidosis, hepatitis, cholecystitis, fungal infectious diseases such as graft-related diseases and immune deficiencies such as vesus-host disease (GVHD) And various inflammatory diseases such as gastric ulcer, tendonitis, and nephritis, and the like.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Pusan University <120> Pharmaceutical composition for immunological conservation          agonists or antagonists of EGFL8 <130> NP12-1252 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> mouse EGFL8 siRNA <400> 1 ggaucuuuca aagagaguu 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EGFL8 forward primer <400> 2 tttcaaagag agtttgggag tg 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGFL8 reverse primer <400> 3 caccacgtgt gtctgtggta 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 forward primer <400> 4 gagagtggac ccaactggaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 reverse primer <400> 5 ctttgggatg gtagctggaa 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> IL-7 forward primer <400> 6 gccctgtcac atcatctgag tgcc 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> IL-7 reverse primer <400> 7 caggaggcat ccaggaactt ctg 23 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GM-CSF forward primer <400> 8 gtcacccggc cttggaagca t 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> GM-CSF reverse primer <400> 9 acagtccgtt tccggagttg g 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TECK forward primer <400> 10 aaactgtggc tttttgcctg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TECK reverse primer <400> 11 cctctggatt cccacacact 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 12 tggagaaacc tgccaagtat g 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 13 ttgtcatacc aggaaatgag c 21

Claims (9)

A T cell-mediated immunosuppressant comprising an EGFL8 (Epidermal growth factor-like protein 8) gene or an expression protein thereof as an active ingredient. The immunosuppressant according to claim 1, wherein the EGFL8 gene is inserted into an expression vector. delete delete delete delete The immunosuppressant according to claim 1, wherein the EGFL8 inhibits the expression of Hes1 and HeIl involved in the intracellular Notch signaling pathway.
delete delete

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