WO2023233665A1 - Immunocyte activator, and therapeutic agent for immunocyte-related inflammatory diseases - Google Patents

Abstract

Provided are: an immunocyte activator containing, as an active ingredient, a substance which inhibits the binding of fibronectin and an immunosuppressive receptor LILRB4; and a therapeutic agent for immunocyte-related inflammatory diseases, the therapeutic agent containing, as an active ingredient, a substance which inhibits the binding of fibronectin and an immunosuppressive receptor LILRB4.

Description

Immune cell activator and therapeutic agent for immune cell-related inflammatory diseases

The present invention relates to an activator for immune cells and a therapeutic agent for immune cell-related inflammatory diseases.

It has been reported that LILRB4 (Leukocyte Ig-like receptor B4, hereinafter also referred to as B4), which is an immunosuppressive receptor, can determine inflammatory diseases derived from infection or autoimmunity ( Patent Document 1).

Furthermore, the physiological ligand of B4 is fibronectin, and it has been reported that a substance that inhibits the binding of B4 and fibronectin is effective in treating immune checkpoint-related diseases (Patent Document 2).

Fibronectin (hereinafter also referred to as FN) is a glycoprotein of approximately 259 kDa that exists in the extracellular matrix (hereinafter also referred to as ECM), cell surfaces, and body fluids. Fibronectin is divided into six regions (domains) by treatment with thermolysin, a proteolytic enzyme. These are based on their specific molecular binding ability. Fibrin/Heparin binding region (Fibrin/Heparin binding-FN), 2. Collagen binding region (Collagen binding-FN), 3. Heparin binding region (Heparin binding-FN), 4. cell/integrin-binding-domain (CBD)-FN; 5. 6. second heparin binding region (second Heparin binding-FN); It is called the second fibrin binding region (second fibrin binding-FN). Thus, FN is composed of multiple domains that differ in their ability to bind to physiological molecules. Until now, in some autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), fluctuations in FN concentration in plasma and body fluids (e.g. joint fluid) have been observed. It has been reported that domain analysis that evaluates FN fragmentation using monoclonal antibodies is useful for diagnosing or evaluating the severity of diseases (Non-Patent Documents 1 to 3). In particular, the Fibrin/Heparin binding-FN concentration was 24±12μg/ml (p<0.003) for SLE and 36±22μg/ml (p<0.003) for RA compared to healthy subjects (61±18μg/ml). .00002) and is expected to be used for diagnosis. Furthermore, it has been reported that FN promotes the metastatic ability and invasive ability of lung cancer cell lines (Non-Patent Document 4).

Various cells within a living body maintain homeostasis while exerting functions such as activation, proliferation, migration, and migration as necessary. In order to maintain this homeostasis and function, cells have focal adhesion with extracellular matrices and other molecules such as collagen, fibronectin, laminin, and proteoglycans on the surfaces of cells and tissues in the surrounding environment. It is necessary to form localized structures called plaques, adhere to cells, and introduce signals into their own cells.
The receptors mainly used for the formation of this focal adhesion are a group of molecules collectively called integrins (hereinafter also referred to as ITG), and the most upstream part of the signal introduced into the cell is two types of tyrosine kinases. Activation by phosphorylation of focal adhesion kinase (FAK), which induces reorganization of actin filaments, and activation by phosphorylation of Spleen tyrosine kinase (Syk), which has the ability to induce inflammation. It has been reported (Non-patent Document 5).

ITG is known to be involved in cell growth, signaling, and cytokine secretion (Non-Patent Document 6). It has been reported that FN as a B4 ligand binds to ITG, and that in the structure of FN, it is RGD in the middle part of FN that binds to ITG (Non-Patent Document 7), and that the α _V of ITG binds to ITG. It has been reported that the _β3 chain is a ligand for mouse B4, and that the binding of ITG and B4 suppresses the activity of mouse bone marrow-derived mast cells BMMC (Non-Patent Document 8).

In addition, amyloid β excreted from microglial cells, which are one of the immune cells, is known to accumulate in the brain and cause chronic inflammation, causing dementia. It has been reported that it may be a cause of chronic inflammation within the body (Non-patent Documents 9 to 11).

Up to now, no molecules are known that suppress signals after the formation of focal adhesions such as ITG. Therefore, many examples of drug discovery targeting cancer and the like that target the ITG molecule itself are known, but drug discovery that targets molecules that suppress ITG is not known. Furthermore, the role of B4 in signal transduction of immune cells including microglial cells is unknown.

Japanese Patent Application Publication No. 2018-25554 International Publication No. 2021/029318

The purpose of the present invention is to provide an activator for immune cells and a therapeutic agent for immune cell-related inflammatory diseases.

The present inventors demonstrated that activation of immune cells caused by fibronectin binding to integrin on the surface of immune cells is suppressed by binding of fibronectin to LILRB4 on the surface of immune cells. I found out. Based on this finding, we discovered that a substance that inhibits the binding between fibronectin and LILRB4 can release the inhibition of immune cell activation caused by fibronectin by LILRB4, activate immune cells, and induce inflammation-inducing effects in immune cells. The present invention was completed based on the discovery that a substance that inhibits the binding between LILRB4 and fibronectin is useful as an activator for immune cells and a therapeutic agent for immune cell-related inflammatory diseases.
The present invention includes the following aspects.
[1] An activator for immune cells containing as an active ingredient a substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4.
[2] The immune cell activator according to [1], wherein the activation of the immune cells is an inflammation-inducing effect of the immune cells.
[3] The activity of the immune cell according to [2], wherein the inflammatory action of the immune cell is due to release of suppression by the immunosuppressive receptor LILRB4 against the inflammatory action of the immune cell based on integrin activation. agent.
[4] The immune cell activator according to [3], wherein the activation of the integrin is due to binding of fibronectin to the integrin.
[5] The immune cell according to any one of [1] to [4], wherein the fibronectin binds to the immunosuppressive receptor LILRB4 via the amino acid sequence shown by SEQ ID NO: 1 in the fibronectin. activator.
[6] The substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4 is an anti-fibronectin antibody or a derivative thereof, an anti-immunosuppressive receptor LILRB4 antibody or a derivative thereof, or a fibronectin analog [1] The immune cell activator according to any one of [5].
[7] The immune cell activator according to [6], wherein the anti-fibronectin antibody or its derivative binds to the amino acid sequence shown by SEQ ID NO: 1 in fibronectin.
[8] The immune cell activator according to [6], wherein the fibronectin analog is any one of the following peptides (a) to (c).
(a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) Contains an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and has a binding site for fibronectin of the immunosuppressive receptor LILRB4. a peptide with binding ability,
(c) A peptide comprising an amino acid sequence having 80% or more identity in the amino acid sequence represented by SEQ ID NO: 1, and having the ability to bind to the fibronectin binding site of the immunosuppressive receptor LILRB4 [9] Said The immune cell activator according to any one of [1] to [8], wherein the immune cell is selected from the group consisting of macrophages, microglial cells, dendritic cells, and NK cells.
[10] A therapeutic agent for immune cell-related inflammatory diseases containing as an active ingredient a substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4.
[11] The therapeutic agent according to [10], wherein the fibronectin binds to the immunosuppressive receptor LILRB4 via the amino acid sequence represented by SEQ ID NO: 1 in the fibronectin.
[12] The substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4 is an anti-fibronectin antibody or a derivative thereof, an anti-immunosuppressive receptor LILRB4 antibody or a derivative thereof, or a fibronectin analog, [10] or The therapeutic agent according to [11].
[13] The therapeutic agent according to [12], wherein the anti-fibronectin antibody or its derivative binds to the amino acid sequence represented by SEQ ID NO: 1 in fibronectin.
[14] The therapeutic agent according to [12], wherein the fibronectin analog is any one of the following peptides (a) to (c).
(a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) Contains an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and has a binding site for fibronectin of the immunosuppressive receptor LILRB4. a peptide with binding ability,
(c) A peptide comprising an amino acid sequence having 80% or more identity in the amino acid sequence represented by SEQ ID NO: 1, and having the ability to bind to the fibronectin binding site of the immunosuppressive receptor LILRB4 [15] Said The therapeutic agent according to any one of [10] to [14], wherein the immune cells are selected from the group consisting of macrophages, microglial cells, dendritic cells, and NK cells.
[16] The immune cell-related inflammatory disease is selected from the group consisting of neurodegenerative diseases, mental developmental disorders, schizophrenia, autism spectrum disorders, atherosclerosis, and infectious diseases, [10] The therapeutic agent according to any one of [15].
[17] The therapeutic agent according to [16], wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, dementia, cerebral infarction, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

According to the present invention, an agent for activating immune cells and a therapeutic agent for immune cell-related inflammatory diseases can be provided.

FIG. 2 is a diagram showing the mechanism of activation of immune cells and the pro-inflammatory effect by fibronectin and integrins, and the suppression of the activation and pro-inflammatory effect of immune cells by LILRB4. FIG. 2 is a diagram showing the results of co-immunoprecipitation assay in Example 1. Intraperitoneal resident F4/80 ^hi macrophages of wild-type mice and gp48-deficient mice, thioglycollate-induced peritoneal F4/80 ^hi macrophages, splenic F4/80 ^hi macrophages, mouse macrophage cell line RAW264.7, and human monocytes FIG. 2 is a diagram showing the results of examining the expression profile of LILRB4/gp49, integrin, and fibronectin in cell line THP-1 by flow cytometry analysis. WT indicates a wild-type mouse, and KO indicates a gp48-deficient mouse. The results of examining the expression of gp49 on mouse macrophage cell line RAW264.7 cells and gp49B-deficient mouse macrophage cell line RAW264.7 cells by flow cytometry are shown. Fig. 2 is a diagram showing the results of examining the confocal signal correlation between gp49, fibronectin, and integrin β ₁ chain on fibronectin-binding peritoneal resident macrophages using confocal laser scanning microscopy analysis, and calculating Pearson's correlation coefficient. . The signal correlation between gp49, fibronectin, and integrin β ₁ chain on fibronectin-binding peritoneal resident macrophages was investigated using confocal laser scanning microscopy analysis, and the results of calculating Pearson's correlation coefficient and Mandel's correlation coefficient are shown. This is a diagram. These are micrographs showing the adhesion of induced peritoneal macrophages to fibronectin-coated or non-coated glass coverslips observed over time. This is a graph obtained by randomly selecting cells, digitizing micrographs of each cell, and calculating the vertical and horizontal lengths of induced peritoneal macrophages adhered to fibronectin-coated or uncoated glass coverslips. . Graphs are shown comparing the cell size of induced peritoneal macrophages adhered to fibronectin-coated or uncoated glass coverslips, respectively. FIG. 3 is a diagram showing the correlation between the confocal signal of integrin β ₁ and the confocal signal of gp49 on non-fibronectin tethering-induced macrophages adhered to fibronectin-uncoated or fibronectin-coated glass using Pearson's correlation coefficient. Results of western blotting analysis of tyrosine phosphorylation of FAK and Syk in fibronectin-coated culture dishes or non-fibronectin-coated culture dishes of wild-type human monocytic cell line THP-1 cells and LILRB4-deficient human monocytic cell line THP-1 cells. FIG. The upper figure shows Western blotting of phosphorylated FAK and total FAK, and the lower figure shows Western blotting of phosphorylated Syk and total Syk, respectively. The results of Western blotting analysis of tyrosine phosphorylation of FAK and Syk in fibronectin-coated culture dishes or non-fibronectin-coated culture dishes of wild-type mouse macrophage cell line RAW264.7 cells and LILRB4-deficient mouse macrophage cell line RAW264.7 cells are shown. This is a diagram. The upper figure shows Western blotting of phosphorylated FAK and total FAK, and the lower figure shows Western blotting of phosphorylated Syk and total Syk, respectively. WT indicates wild-type cells, and KO indicates LILRB4-deficient cells. FIG. 2 is a diagram showing the gating process of CD11c ⁺ MHC-II ^high bone marrow-derived cultured dendritic cells (BMDC) for flow cytometric analysis of cell surface expression of gp49, integrin, and fibronectin. FSC indicates forward scattering, SSC indicates side scattering, A indicates area, H indicates height, and PE indicates phycoerythrin. The PE profile represents a base level for further analysis. The CD11c ⁺ MHC-II ^high BMDCs obtained by the gating in FIG. 10A were isolated as cells that were floating and had low adhesion and were referred to as MHC class-II high population (left figure), and The remaining adherent CD11c ⁺ MHC-II ^high BMDCs obtained by gating in Figure 10A were also separated and isolated as cells termed MHC class-II low population (right panel). be. Photographs showing the appearance of floating non-adherent BMDCs on a glass cover glass (maximum magnification x 400, left image) and adherent BMDCs on a plastic dish (maximum magnification x 200, right image) under an optical microscope. It is. Arrows indicate typical CD11c ⁺ MHCII ^high BMDCs with many dendrites. FIG. 3 is a diagram showing the gating process of spleen CD3 ⁻ CD19 ⁻ CD11c ⁺ MHC-II ^high dendritic cells (DC) in flow cytometry analysis of cell surface expression of gp49, integrin, and fibronectin. FIG. 2 is a diagram showing cell surface expression of gp49, integrin α and β chains, FN30, and FN module III (FNIII) analyzed by flow cytometry of mouse bone marrow-derived dendritic cells BMDC and spleen dendritic cells (DC). A: Cells expressing gp49A and gp49B in MHC class-II high population (left) and MHC class-II low population (right) by flow cytometry to determine the expression level of gp49B in mouse BMDC. FIG. 3 is a diagram showing the results of analysis of surface expression. WT indicates BMDCs isolated from a wild-type mouse, and gp49BKO indicates BMDCs isolated from a gp49B-deficient mouse. B shows the results of analyzing mouse peritoneal macrophages as a positive control and BMDC for cell surface expression of FN30 and FNIII by flow cytometry. FIG. 2 is a diagram showing the correlation between gp49B and integrin β ₁ signals on BMDC and the correlation between the signals of MHC class I α chain and β ₂ microglobulin (β ₂ m) as a positive control. FIG. 3 shows Western blot analysis of tyrosine phosphorylation of FAK after stimulation of BMDCs prepared from wild-type or gp49B-deficient mice with plate-bound immobilized fibronectin (FN). The upper figure shows phosphorylated FAK, and the lower figure shows total FAK. FIG. 3 shows Western blot analysis of tyrosine phosphorylated Syk after stimulation of BMDCs isolated from wild-type mice or gp49B-deficient mice with immobilized FN. The upper figure shows phosphorylated Syk, and the lower figure shows total Syk. BMDC WT indicates BMDC prepared from a wild-type mouse, and BMDC gp49B ^−/− indicates BMDC prepared from a gp48-deficient mouse. Integrin β ₁ and gp49B on the surface of mouse NK cells were stained with fluorescently labeled antibodies, observed with a confocal laser scanning fluorescence microscope, the obtained confocal signals were digitized, and the Pearson coefficient between signal intensities was calculated. FIG. This is a photograph in which microglial cells isolated from wild-type mice or gp49B gene-deficient mice were seeded onto FN-coated plates, cultured, and the morphology of the cells was observed using a microscope. 1 is a graph showing the amount of inflammatory cytokine TNF-α secreted from microglial cells isolated from wild-type mice or gp49B gene-deficient mice. FIG. 3 is a diagram showing the results of measuring the long axis length of each microglial cell isolated from a wild-type mouse or a gp49B gene-deficient mouse. WT indicates the results of microglial cells isolated from wild-type mice, and KO indicates the results of microglial cells isolated from gp49B gene-deficient mice.

[Immune cell activator]
The immune cell activator of the present invention contains a substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4 as an active ingredient. Fibronectin can bind to LILRB4 via the amino acid sequence represented by SEQ ID NO: 1 in the fibronectin. That is, the amino acid sequence represented by SEQ ID NO: 1 is the target sequence of LILRB4 in fibronectin.
Substances that inhibit the binding between fibronectin and LILRB4 are not particularly limited as long as they have the activity of inhibiting the binding between fibronectin and LILRB4, but anti-fibronectin antibodies or derivatives thereof, anti-LILRB4 antibodies or derivatives thereof, fibronectin Examples include analog.

The anti-fibronectin antibody may be either a monoclonal antibody or a polyclonal antibody as long as it reacts with fibronectin, but monoclonal antibodies are preferably used. The antibody can be produced by a well-known method. For example, in the production of polyclonal antibodies, mice, rats, hamsters, rabbits, goats, sheep, chickens, and the like are used as animals to be immunized. Antiserum can be obtained from serum after the antigen is administered subcutaneously, intradermally, intraperitoneally, etc. to an animal once or multiple times. When using proteins or peptides as antigens, it is more preferable to immunize with a mixture with a replacement fluid that has an immunostimulatory effect.

In addition, for the production of monoclonal antibodies, known methods for producing monoclonal antibodies can be used, for example, "Monoclonal Antibodies" co-authored by Kaoaki Nagasune and Hiroshi Terada, Hirokawa Shoten (1990), and by James W. Golding, "Monoclonal Antibody", 3rd edition, Academic Press, 1996. Monoclonal antibodies can also be produced by DNA immunization, as described in Nature 1992 Mar 12; 356 152-154 and J. It can be produced with reference to Immunol Methods Mar 1; 249 147-154.

As the antigen used for antibody production, fibronectin or a partial fragment thereof (peptide), or a vector incorporating cDNA encoding fibronectin or a partial fragment thereof can be used. In order to obtain a monoclonal antibody that inhibits the binding between fibronectin and LILRB4, it is preferable to use a peptide containing the amino acid sequence represented by SEQ ID NO: 1, which is the target sequence of LILRB4 in fibronectin. A fibronectin vector, which is a construct containing a gene encoding a peptide containing the amino acid sequence represented by SEQ ID NO: 1, can be used as an optimal antigen gene for immunization. In the DNA immunization method, the above-mentioned gene constructs are introduced into an animal (mouse, or This can be carried out by subcutaneously injecting it into a rat, etc.) and allowing it to be taken up into the cells.

The anti-fibronectin monoclonal antibody can be produced by culturing a hybridoma prepared according to a conventional method and separating it from the culture supernatant, or by administering the hybridoma to a compatible mammal and collecting it as ascites fluid. Furthermore, the anti-fibronectin monoclonal antibody can also be produced using known genetic recombination techniques. Specifically, the monoclonal antibody produced by the hybridoma prepared above and the gene encoding the antibody are cloned, a vector containing the gene is prepared, and this is introduced into a host cell and transformed. It can be produced by obtaining cells expressing fibronectin antibodies and culturing them. The cells, vector types, cell types, culture conditions, etc. used for this preparation are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.

The antibody can be further purified and used if necessary. Methods for purifying and isolating antibodies include conventionally known methods, such as salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion exchange chromatography, and affinity purification using protein A columns. It will be done.

Examples of derivatives of anti-fibronectin antibodies include F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusions containing the antibody portion of the anti-fibronectin antibody. Examples include peptides. Anti-fibronectin antibody derivatives can be produced according to known methods for producing antibody derivatives.

The anti-fibronectin antibody or its derivative binds to the amino acid sequence represented by SEQ ID NO: 1 in fibronectin or a partial sequence thereof. In the immune cell activator of the present invention, the anti-fibronectin antibody or its derivative inhibits the binding between fibronectin and LILRB4 by binding to the amino acid sequence represented by SEQ ID NO: 1 in fibronectin or a partial sequence thereof. be able to.

The anti-LILRB4 antibody may be either a monoclonal antibody or a polyclonal antibody as long as it binds to LILRB4, but monoclonal antibodies are preferably used. The anti-LILRB4 antibody can be produced by the same method as the anti-fibronectin antibody.

As the antigen used for anti-LILRB4 antibody production, a vector incorporating the LILRB4 protein, a partial fragment thereof (peptide), or a cDNA encoding the LILRB4 protein can be used. In order to obtain a monoclonal antibody that recognizes the higher-order structure of LILRB4, a full-length LILRB4 vector, which is a construct containing the full-length human LILRB4 gene, is the optimal antigen gene for immunization. These constructs can also be used as antigen genes for immunization. The partial region of the LILRB4 sequence is preferably a region of LILRB4 (fibronectin binding site) where the amino acid sequence represented by SEQ ID NO: 1, which is the target sequence of LILRB4 in fibronectin, binds to LILRB4. In the DNA immunization method, the above-mentioned gene constructs are introduced into an animal (mouse, or This can be carried out by subcutaneously injecting it into a rat, etc.) and allowing it to be taken up into the cells.

The anti-LILRB4 antibody can be purified by the same method as the anti-fibronectin antibody. The derivatives of the anti-LILRB4 antibody include, for example, F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, variants thereof, fusion proteins containing the antibody portion of the anti-LILRB4 antibody, or Examples include fusion peptides.

In the immune cell activator of the present invention, the anti-LILRB4 antibody or its derivative can inhibit fibronectin from binding to LILRB4 via the amino acid sequence represented by SEQ ID NO: 1 in fibronectin.

Inhibition of binding between fibronectin and LILRB4 can be performed by evaluating inhibition of binding of fibronectin to cells expressing LILRB4. Cells expressing LILRB4 are not particularly limited as long as they express LILRB4, but include, for example, spleen cells, peripheral blood leukocytes, bone marrow cells, brain cells, or B cells isolated therefrom. , plasma cells, monocytes/macrophages, dendritic cells, eosinophils, basophils, neutrophils, mast cells, activated T cells, microglial cells, and the like.

The nucleotide sequence and amino acid sequence of LILRB4 can be found from the database provided by the National Center for Biotechnology Information (NCBI). In the case of human (Homo sapiens) LILRB4, for example, Entrez Gene ID is 11006 (as of June 17, 2019), RefSeq Protein ID is NP_001265355.2, NP_001265356.2, NP_001 265357.2, NP_001265358.2, NP_001265359.2 (isoform 1 -5). For mouse (Mus musculus) LILRB4, Gene ID is 14728 (as of June 24, 2016) and NP_038560.1, and for rat (Rattus norvegicus) LILRB4, Gene ID is 292594 (as of April 18, 2019). (as of date) , RefSeq Protein ID is NP_001013916, and other animals are also known to have LILRB4. The LILRB4 is not limited to the above LILRB4, and other LILRB4 are also included in the LILRB4 in the present invention.

The base sequence and amino acid sequence of fibronectin can be found from the database provided by the National Center for Biotechnology Information (NCBI). In the case of human (Homo sapiens) fibronectin, for example, Entrez Gene ID is 2335, RefSeq Protein ID is NP_997647, NP_001352447, XP_005246463, etc. For mouse (Mus musculus) fibronectin, Gene ID is 14268, and for rat (Rattus norvegicus) fibronectin, Gene ID is 25661 and RefSeq Protein ID is NP_062016. known to have bronectin . The fibronectin in the present invention is not limited to the above-mentioned fibronectin, and other fibronectins are also included in the fibronectin in the present invention.

Fibronectin activates immune cells by releasing LILRB4-induced suppression of immune cell activation based on ITG activation. The mechanism is shown in Figure 1.
Fibronectin binds to the β chain of ITG primarily through the RGD sequence in fibronectin. When fibronectin binds to ITG via the RGD sequence in fibronectin, phosphorylation of Spleen tyrosine kinase (hereinafter referred to as Syk) and focal adhesion kinase (hereinafter referred to as FAK) downstream of ITG is induced. Ru. When FAK is phosphorylated, it induces actin filament reorganization and functions such as cell adhesion, cell growth, and migration. On the other hand, when Syk is phosphorylated, proinflammatory effects such as secretion of inflammatory cytokines are induced.

Fibronectin binds to LILRB4 present on the cell surface of immune cells via the amino acid sequence represented by SEQ ID NO: 1 in fibronectin (hereinafter also referred to as FN30). When fibronectin binds to LILRB4 via FN30, the immunoreceptor inhibitory effect of LILRB4 is induced by Src family kinases (hereinafter referred to as SFKs) by the same mechanism that phosphorylates Syk when fibronectin binds. The tyrosine motif (ITIM) is phosphorylated, and the proinflammatory effect caused by Syk phosphorylation is suppressed through the action of dephosphorylating enzymes such as SHP-1.
Substances that inhibit the binding of fibronectin to LILRB4 release the suppression by fibronectin of the proinflammatory effect caused by Syk phosphorylation induced by fibronectin binding to ITG by inhibiting the binding of fibronectin to LILRB4. By doing so, it induces the pro-inflammatory action of immune cells. In other words, substances that inhibit the binding of fibronectin to LILRB4 inhibit the binding of fibronectin to LILRB4 through FN30 in fibronectin, thereby inhibiting the binding of fibronectin to ITG through the RGD sequence of fibronectin. Activation of ITG activates immune cells and induces inflammatory effects of immune cells.

As mentioned above, fibronectin activates ITG present on the surface of immune cells, but as immune cells adhere to other cells, tissues, and organ walls, fibronectin-binding properties can be activated in immune cells even in the absence of fibronectin. ITG can be activated non-specifically to ITG or other ITGs. Therefore, the substance of the present invention that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4 can activate immune cells even in the absence of fibronectin.

Furthermore, when immune cells adhere, LILB4 present on the surface of the immune cells comes into close proximity with ITG, thereby suppressing activation of the immune cells by LILRB4 of ITG. Therefore, substances that inhibit the binding between fibronectin and the immunosuppressive receptor LILRB4 can activate immune cells by releasing the suppression of immune cells by LILRB4 even in the absence of fibronectin. can.

In the present invention, immune cells are not particularly limited as long as they are cells involved in immunity; examples include myeloid cells such as macrophages, microglia cells, and dendritic cells; lymphoid cells such as NK cells, T cells, and B cells. Cells include monocytes, granulocytes, mast cells, basophils, etc., but macrophages, microglial cells, dendritic cells, and NK cells are preferred.

In the present invention, the fibronectin analog includes any fibronectin analog as long as it has the effect of inhibiting the binding between fibronectin and LILRB4, and for example, any of the following (a) to (c). Two peptides are mentioned.
(a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) Contains an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and has a binding site for fibronectin of the immunosuppressive receptor LILRB4. a peptide with binding ability,
(c) A peptide that includes an amino acid sequence having 80% or more identity in the amino acid sequence represented by SEQ ID NO: 1 and has the ability to bind to the fibronectin binding site of the immunosuppressive receptor LILRB4.

The identity of the amino acid sequence is 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more. Furthermore, the number of deletions, substitutions, or additions in the above amino acid sequence is preferably 1 to 5, more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2. Amino acid sequence identity can be determined by BLAST searches provided on the GenBank database.

The fibronectin analog may be a fibronectin analog obtained by fusing any one of the peptides (a) to (c) with the Fc region of immunoglobulin G.

The fibronectin analog can be produced by a known method, for example, by genetic recombination technology.

The immune cell activator of the present invention contains a substance that inhibits the binding between fibronectin and LILRB4 as an active ingredient, and may further contain a pharmaceutically acceptable carrier and additives.

Examples of carriers and additives include water, saline, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, Sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol , stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants, and the like, but are not limited to these.

The immune cell activator of the present invention can be in various forms, such as a liquid (for example, an injection), a dispersion, a suspension, a tablet, a pill, a powder, a suppository, and the like. A preferred embodiment is an injection, which is preferably administered parenterally (eg, intravenously, transdermally, intraperitoneally, intramuscularly).

The immune cell activator of the present invention can be used as a therapeutic agent for immune cell-related inflammatory diseases.
The dosage for activating immune cells of the present invention is, for example, 0.025 to 50 mg/kg, preferably 0.1 to 50 mg/kg, more preferably 0.1 to 25 mg/kg, even more preferably 0. .1 to 10 mg/kg or 0.1 to 3 mg/kg, but is not limited thereto.

[Treatment agent for immune cell-related inflammatory diseases]
The therapeutic agent for immune cell-related inflammatory diseases of the present invention contains a substance that inhibits the binding between fibronectin and LILRB4 as an active ingredient.
Substances that inhibit the binding between fibronectin and LILRB4 are not particularly limited as long as they have the activity of inhibiting the binding between fibronectin and LILRB4, but anti-fibronectin antibodies or derivatives thereof, anti-LILRB4 antibodies or derivatives thereof, fibronectin Examples include analog. Anti-fibronectin antibodies or derivatives thereof, anti-LILRB4 antibodies or derivatives thereof, and fibronectin analogs include those described above.

In the present invention, immune cell-related inflammatory diseases are not particularly limited as long as they are inflammatory diseases in which immune cells are involved, but examples include neurodegenerative diseases, mental developmental disorders, schizophrenia, autism spectrum disorders, and chyme. Examples include arteriosclerosis and infectious diseases.

Neurodegenerative diseases are not particularly limited as long as they are caused by neurodegeneration, but include Alzheimer's disease, dementia, cerebral infarction, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and the like.

As shown in Figure 1, fibronectin binds to the β chain of ITG through the RGD sequence in fibronectin. When fibronectin binds to ITG via the RGD sequence in fibronectin, phosphorylation of Syk and FAK downstream of ITG is induced. When FAK is phosphorylated, it induces actin filament reorganization and functions such as cell adhesion, cell growth, and migration. On the other hand, when Syk is phosphorylated, proinflammatory effects such as secretion of inflammatory cytokines are induced.

Fibronectin binds to LILRB4 present on the cell surface of immune cells via FN30 represented by SEQ ID NO: 1 in fibronectin. When fibronectin binds to LILRB4 via FN30, the binding of fibronectin activates ITG, which activates SFK, phosphorylates the immunoreceptor inhibitory tyrosine motif (ITIM) of LILRB4, and phosphorylates Syk. suppresses the inflammatory effects caused by
Substances that inhibit the binding of fibronectin to LILRB4 release the suppression by fibronectin of the proinflammatory effect caused by Syk phosphorylation induced by fibronectin binding to ITG by inhibiting the binding of fibronectin to LILRB4. By doing so, the pro-inflammatory effect of immune cells is induced, and the pro-inflammatory effect such as secretion of inflammatory cytokines is induced. In other words, substances that inhibit the binding of fibronectin to LILRB4 inhibit the binding of fibronectin to LILRB4 through FN30 in fibronectin, thereby inhibiting the binding of fibronectin to ITG through the RGD sequence of fibronectin. Activation of ITG induces pro-inflammatory effects.

When immune cells are activated and their pro-inflammatory action is induced, for example, amyloid-β is phagocytosed by immune cells in the brain, and accumulation of amyloid-β in the brain is suppressed, leading to Alzheimer's disease. It can treat neurodegenerative diseases such as dementia and dementia.
In addition, in atherosclerosis, macrophages accumulate in vascular endothelial cells in an attempt to phagocytose cholesterol, etc., but they are unable to sufficiently remove this and become foamy macrophages that remain locally aggregated. The lumen of blood vessels narrows, causing atherosclerosis. By releasing the suppression of LILRB4 by fibronectin and activating macrophages, the formation of macrophages is prevented, and the activated macrophages remove cholesterol and prevent narrowing of the vascular lumen, resulting in atherosclerosis. Can treat sclerosis.
In addition, in chronic infections, pathogenic bacteria may not be sufficiently removed by immune cells such as macrophages, or inflamed tissue may not be sufficiently repaired, resulting in a prolonged chronic inflammatory state. In such cases, by activating immune cells such as macrophages, chronic inflammation is relieved and sufficient inflammation is induced to achieve complete removal of pathogens and sufficient tissue repair. can treat infections.

As mentioned above, fibronectin activates ITG present on the surface of immune cells, but due to adhesion of immune cells, even in the absence of fibronectin, immune cells activate non-specific ITGs that bind to fibronectin and other ITGs. can activate ITG in immune cells and induce pro-inflammatory effects. Therefore, the substance of the present invention that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4 can induce the proinflammatory action of immune cells even in the absence of fibronectin.

Furthermore, when immune cells adhere, LILB4 present on the surface of the immune cells comes into a close relationship with ITG, thereby suppressing the activation of the immune cells by LILRB4 of ITG and suppressing the inflammation-inducing effect of the immune cells. Therefore, a substance that inhibits the binding of fibronectin to the immunosuppressive receptor LILRB4 releases the suppression of immune cells by LILRB4 in immune cells even in the absence of fibronectin, activates immune cells, and exerts proinflammatory effects. By inducing immune cells, it is possible to treat immune cell-related inflammatory diseases.

In the therapeutic agent for immune-related inflammatory diseases of the present invention, the immune cells are not particularly limited as long as they are cells involved in immunity; for example, myeloid cells such as macrophages, microglial cells, and dendritic cells; NK cells; , T cells, B cells, and other lymphoid cells; monocytes, granulocytes, mast cells, basophils, etc., but macrophages, microglial cells, dendritic cells, and NK cells are preferred.

The therapeutic agent for immune cell-related inflammatory diseases of the present invention may further contain pharmaceutically acceptable carriers and additives. Pharmaceutically acceptable carriers and additives include those mentioned above.

The therapeutic agent for immune cell-related inflammatory diseases of the present invention can be in various forms, such as solutions (eg, injections), dispersions, suspensions, tablets, pills, powders, suppositories, and the like. A preferred embodiment is an injection, which is preferably administered parenterally (eg, intravenously, transdermally, intraperitoneally, intramuscularly).

The dosage of the therapeutic agent for immune cell-related inflammatory diseases of the present invention is, for example, 0.025 to 50 mg/kg, preferably 0.1 to 50 mg/kg, more preferably 0.1 to 25 mg/kg, and It can be preferably 0.1 to 10 mg/kg or 0.1 to 3 mg/kg, but is not limited thereto.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Formation of LILRB4, fibronectin, and integrin complexes in macrophages A possible complex between gp49B (LILRB4 in mice), fibronectin (hereinafter referred to as FN), and integrins in bone marrow-derived macrophages was determined by co-immunoprecipitation assay. Body formation was monitored.
Co-immunoprecipitation assay was performed by the following method.
Bone marrow cells derived from C57BL/6 (B6) mice (manufactured by CLEA) were isolated for differentiation of bone marrow-derived macrophages (BMM) based on a previous report (Weischenfeldt & Porse 2018; doi: 10.1101/pdb.prot5080). .
The obtained bone marrow cells (2 × 10 ⁷ cells) were seeded in a 10 cm dish and cultured for 6 days in α-MEM medium supplemented with 10% FCS and 20 ng/ml M-CSF, and then mature BMM was lysed in a non-denaturing manner. It was dissolved in buffer (20mM Tris-HCl pH 8, 137mM NaCl, 1% Nonidet P-40 and 2mM EDTA). Cell lysate (300 μg/reaction solution) was incubated with 2 μg of anti-FN antibody (F3648, manufactured by Sigma-Aldrich) or control IgG (manufactured by Santa Cruz), and the protein-antibody complex was incubated with Dynabeads protein G (Thermo Fisher). (manufactured by Scientific). The obtained immune complex was resuspended in 1x SDS sample buffer, and anti-ILT3 antibody (ab231813, manufactured by Abcam), anti-FN antibody (F3648, manufactured by Sigma-Aldrich), and anti-integrin β ₁ antibody (GTX128839) were added. , manufactured by GeneTex) for Western blotting analysis.
The results of the co-immunoprecipitation assay are shown in Figure 2. As shown in FIG. 2, the immunoprecipitated sample with anti-FN antibody contained integrin β ₁ and gp49B, suggesting a direct or indirect association of the three molecules of FN, integrin β ₁ , and gp49B.

Next, to verify the formation of a complex (triplet) of gp49B, FN, and integrin, we examined mouse intraperitoneal resident F4/80 ^hi macrophages, thioglycollate-induced peritoneal F4/80 ^hi macrophages, splenic F4/80 ^hi macrophages, Expression profiles of LILRB4/gp49B, FN-binding integrin subunits, and FN on the surface of primary cultured macrophages, monocytic cell lines, and macrophage cell lines, including the mouse macrophage cell line RAW264.7 and the human monocytic cell line THP-1. I looked into it. Isolation of primary macrophages from mouse tissues was performed by magnetic cell sorting and/or flow cytometric sorting. The mice used were wild-type mice and gp48-deficient mice prepared based on a previous report (Kasai S et al. Eur J Immunol 2008;38:2426-2437).

First, peritoneal induced macrophages with a purity of 90% or more were collected. These cells were incubated with anti-LILRB4 antibody (clone ZM4.1, clone #293623; manufactured by Thermo Fisher Scientific), anti-gp49 antibody (manufactured by Santa-Cruz Biotechnology), and anti-integrin subunit _α5 antibody (manufactured by BioLegend). ), Anti-integrin α _V antibody (manufactured by BioLegend), anti-integrin β ₁ antibody (manufactured by Merck Millipore, Thermo Fisher Scientific, Sigma-Aldrich), anti-integrin β ₃ antibody (manufactured by BioLegend), anti-FN 30 antibodies ( WO2021/029318) and stained with anti-FNIII _1-7 antibody (manufactured by Thermo Fisher Scientific). The results are shown in FIG. As shown in FIG. 3, strong expression of integrin α _V , β ₁ and β ₃ chains, gp49, FN30 and FNIII _1-7 was detected in mouse intraperitoneal resident macrophages.

Furthermore, gp49 expression signals were decreased in cells from gp49B-deficient mice. This indicates that the majority of the gp49 expression signal is the gp49B isoform rather than the gp49A. In mouse-induced peritoneal macrophages, expression of integrin α ₅ , α _V and β ₁ chain, and gp49 was detected, but FN30 was not detected and FNIII _1-7 was hardly detected. Furthermore, similar to mouse intraperitoneal resident macrophages, gp49 signals were shown to be mostly derived from gp49B. In mouse splenic F4/80 macrophages, expression of integrin α _V and β ₁ chains was strong, but gp49 signals derived from the gp49B isoform were weak, and neither FN30 nor FNIII _1-7 was detected. In RAW264.7 cells, we detected strong expression of integrin α ₅ , α _V and β ₁ chains, and gp49, but not FN30 or FNIII _1-7 .

Furthermore, in order to determine the expression level of gp48B, the expression of gp49 on mouse macrophage cell line RAW264.7 cells and gp49B-deficient mouse macrophage cell line RAW264.7 cells was examined by flow cytometry. The results are shown in FIG. As shown in Figure 4, the gp49 expression signal was decreased in gp49B-deficient RAW264.7 cells. This indicates that the gp49 signal is mainly a gp49B signal. Furthermore, as shown in FIG. 3, expression of LILRB4, integrin _α5 and _β1 chain was detected on the THP-1 cell surface, but FN30 or FNIII _1-7 was not detected.
These results indicate that LILRB4/gp49B expression is negligible in splenic macrophages, but detected in primary/induced macrophages as well as monocyte/macrophage cell lines. The expression profile of the integrin subunits that constitute the major FN-binding integrins was characteristic for each type of primary cell or cell line. Surface FN was exceptionally strongly detected on peritoneal resident macrophages, but all other cells had little, if any, tethered FN on their surface.

[Example 2] Signal correlation between gp49B, FN, and integrin on macrophages Next, the signal correlation between gp49, FN, and integrin β ₁ chain on FN-bound peritoneal resident macrophages was analyzed using confocal laser scanning microscopy. It was investigated by.
Confocal laser scanning microscopy analysis was performed as follows.
Cells were seeded in a glass bottom dish (manufactured by Matsunami, polylysine coated, dish diameter 35 mm, glass diameter 14 mmφ, glass thickness No. 1S/1.5 (0.16-0.19 mm), #D11131H) and cultured for 1 hour. did. Cells were fixed with 2% paraformaldehyde (PFA) for 1 hour at room temperature. Next, the fixed cells were treated with AlexaFluor546-conjugated anti-gp49 (manufactured by Santa Cruz, #5358A458F546), AlexaFluor647-conjugated anti-integrin β ₁ (manufactured by ThermoFisher Scientific, MAB1997-AF647), Al exaFluor488-conjugated anti-FN30, unconjugated anti-FN (Invitrogen) Co., Ltd., #MA5-29279) and stained with AlexaFluor488 AffiniPure F(ab') ₂ fragment donkey anti-rabbit IgG (H+L). After thoroughly washing the cells with a washing solution (PBS containing 1% BSA), the cells were seeded in a medium containing 35 g glycerol in 100 ml of PBS, and then subjected to a Leica SP8, Olympus FV3000, or Nikon A1 confocal microscope. Tested in 2-D under the system. Confocal images of cells were also analyzed by SP8, FV3000 or Nikon A-1 3D (z-axis) analysis software. The statistical analysis was performed using GraphPad Prism® 6 (Version 6.0; manufactured by GraphPad Software) or R software (ver 4.1.0, manufactured by Lucent Technologies), and was performed using at least three independent experiments. Based on the results. Data were expressed as mean ± SEM or ±SD. Data were compared for statistical differences on Pearson's correlation coefficient using a two-tailed test, two-tailed paired Student's t-test. p<0.05 was considered statistically significant.

The results of statistical analysis of the confocal laser scanning microscopy analysis results are shown in FIG. As shown in Figure 5, confocal images of cell contours showed that all combinations, namely FN30-gp49 (r=0.231±0.096), gp49-integrin _β1 (r=0.356±0 .088) and integrin β ₁ -FN30 (r=0.320±0.093).
To confirm the above observations, we further analyzed the correlation values in two sets of experiments using different confocal systems. The results are shown in FIG. As shown in FIG. 6, the Pearson and Mandel r values were 0.235 to 0.543, and all combinations showed positive correlation. These results indicate that some (but not all) of gp49B, FN30, and integrin _β1 are spatially close to each other, and as shown in Fig. 1, gp49B-FN-integrin on FN-bound macrophages. This shows that a trimeric complex is formed.

[Example 3] Regarding the effect of immobilized FN on integrin-gp49 signal correlation Cell adhesion to plastic or glass plates is caused by the interaction between the plastic/glass surface and cell adhesion molecules such as integrins and cell non-adhesion molecules such as gp49B. This is thought to include non-specific interactions. Cell activation is initiated even when adhesion-specific integrins are involved in non-specific adhesion. On the other hand, cell adhesion to FN-rich matrices is mediated by FN-binding integrins. Therefore, we next investigated how immobilized FN affects the adhesion-inducing integrin-gp49 signal correlation. To this end, we first monitored the adhesion of FN-poor peritoneal-induced macrophages to FN-coated and uncoated glass coverslips over time. The results are shown in FIGS. 7A to 7C. Figure 7A is a photograph of microscopic observation over time, Figure 7B is a graph of digitized micrographs of randomly selected cells, and the vertical and horizontal lengths are calculated. Figure 7C is a graph of the cell size calculated by √{( The graph is calculated as (horizontal length) ² + (vertical length) ² } and compares cells adhered to FN-coated or non-coated glass. As shown in FIG. 7A, microscopic observation revealed that macrophages began to adhere to the glass 1 hour after seeding. After overnight culture, many cells showed a typical spindle-like shape. When the area of each cell was measured, it was found that after 1 to 3 hours, more cells on the FN-coated glass exhibited a diffuse shape than on the uncoated glass, as shown in FIG. 7B. Notably, as shown in Figure 7C, at the 1 hour time point, the cell area was significantly larger on the FN-coated glass.
These results suggested that the contribution of FN-bound integrins was predominant in cell adhesion to FN-coated glass for up to 1 hour of culture. From this result, it was inferred that the longer the culture time, the greater the involvement of nonspecific adhesion mediated by various cell surface molecules including gp49B.

Next, in the same manner as in Example 2, after 1 hour of culture, the confocal signal of integrin β ₁ and gp49 on non-FN-tethering-induced macrophages adhered to FN-uncoated glass or FN-coated glass. The correlation with the focal signal was investigated. The focal plane was set at the bottom of the cells adhered to the glass, and signals on the cell outline were analyzed.
As a result, each cell showed an overall circular signal for gp49 and integrin _β1 , and as shown in Figure 8, in the focal plane after 1 hour, gp49- A significant correlation of integrin β ₁ signals was observed (r=0.449±0.160). However, there was no significant difference between cells attached to FN-coated glass and cells attached to uncoated glass. This suggests a greater involvement of nonspecific adhesion of gp49B-containing membrane molecules to focal adhesion sites, even in the absence of exogenous FN, even in the absence of exogenous FN. It was suggested that a significant portion of the fibroblast was located close to the focal adhesion site.

[Example 4]
As shown in Figure 1, cell adhesion following integrin activation directly induces downstream phosphorylation of FAK and Syk by Src family kinases, leading to classical signaling (cell adhesion, growth and migration signals, respectively). transduction), triggering pro-inflammatory signaling. Therefore, we investigated as follows whether phosphorylation of FAK and Syk could be regulated after integrin binding to FN in the presence or absence of LILRB4/gp49B.

FN-negative human monocytic cell line THP-1 cells were seeded onto FN-coated culture dishes, incubated, and cell lysates for Western blot analysis were prepared as in Example 1. Next, control wild-type THP-1 cells and LILRB4-deficient THP-1 cells were stimulated with FN or BSA immobilized on a dish, or an uncoated dish, and tyrosine phosphorylation of FAK or Syk was examined by Western blot analysis. Ta. The results are shown in FIG. 9A. As shown in Figure 9A, in wild-type THP-1 cells, significant tyrosine phosphorylation of Syk was induced when cells adhered to FN-coated dishes, whereas phosphorylation of FAK was less pronounced. . Furthermore, increased Syk phosphorylation was observed in LILRB4-deficient THP-1, suggesting that Syk phosphorylation is negatively regulated by LILRB4.
Furthermore, the phosphorylation of FAK and Syk was examined using Western blotting in the same manner as above using the mouse macrophage cell line RAW264.7 instead of the human monocyte cell line THP-1 cells. The results are shown in FIG. 9B.
As shown in Figure 9B, in the murine macrophage cell line RAW264.7, significant tyrosine phosphorylation of Syk was also induced when cells adhered to FN-coated dishes, but phosphorylation of FAK was less pronounced. There wasn't. Furthermore, increased Syk phosphorylation was observed in LILRB4-deficient RAW264.7, suggesting that Syk phosphorylation is negatively regulated by LILRB4.
These results indicated that BILRB4/gp49B functions as a regulator of Syk-mediated proinflammatory signaling induced by FN-binding integrins.

[Example 5] Expression profile of integrins, FN, and gp49B on bone marrow-derived cultured dendritic cells (BMDCs) Major FN-binding integrins on the surface of mouse BMDCs, namely integrins α ₅ , α _V , β ₁ and β ₃ , In addition, the expression profiles of FN and gp49B subunits were investigated as follows.
First, spleen cells and bone marrow cells were prepared from the spleen, femur, and tibia of C57BL/6 mice, respectively, and red blood cells were dissolved and removed with a solution of 155 mM NH ₄ Cl, 10 mM KHCO ₃ , and 0.1 mM EDTA. To prepare bone marrow-derived cultured dendritic cells (BMDCs), bone marrow cells were incubated with heat-inactivated fetal bovine serum (FBS, manufactured by Biowest), 1mM sodium pyruvate, 50μM 2-mercaptoethanol, and 100U/ml penicillin (Sigma- Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 20 ng/ml GM-CSF (PeproTech) using RPMI1640 (Sigma-Aldrich) at 37°C and 5% _CO2. Cultured in a humidified environment containing On the 4th day, the culture medium was replaced, and on the 6th day, after rotating the culture bottle, floating/low-adhesion cells were collected and used as BMDCs.

The floating/low-adhesive cells were collected by pipetting, sorted into lymphocytes and singlets, and then sorted and purified to contain mainly MHC class II-high CD11c ⁺ cells and few MHC class II-low cells. A population containing the following was obtained (Fig. 10A, Fig. 10B). Hereinafter, this population will be referred to as the class II-high population. In parallel, analysis of non-planktonic and adherent cells on day 6 of culture revealed that most of them were MHC class II-low compared to MHC class II-high planktonic and low-adherent BMDCs. It was revealed that these were immature BMDCs (Fig. 10B). Hereinafter, this population will be referred to as the MHC class II-low population.
As shown in Figure 10C, microscopic observation revealed cells rich in dendrites in the floating/low-adhesion cell population, which are relatively mature BMDCs compared to adherent immature BMDCs. This is a typical characteristic of Furthermore, as shown in FIG. 10D, primary dendritic cells (primary DC) were isolated from splenocytes by selecting CD3 ⁻ CD19 ⁻ CD11c ^high MHC class II ^high cells.

Next, flow cytometry analysis of BMDC was performed for gp49, integrin subunit, FN30, and FNIII type repeat 7-9 (FNIII _7-9 ). The results are shown in FIGS. 11 and 12. As shown in Figure 11, the MHC class II-high population was positive for surface gp49, integrins α ₅ , α _V , and β ₁ but negative for integrin β ₃ , FN30, and FNIII. Met. Expression of gp49 is not necessarily strong, and as shown in FIG. 12, when BMDCs prepared from gp49B-deficient mice were used, gp49B isoform expression was dominant, but gp49A isoform expression was not. From this result, it was concluded that gp49B and FN-binding integrins α ₅ β ₁ and α _V β ₁ are expressed on MHC class II-high BMDCs, but that FN is not constitutively bound to BMDCs.

Similarly, expression of integrin α ₅ , α _V , β ₁ and β ₃ subunits was found in the MHC class II-low population. Note that in the MHC class II-low population, integrin β ₁ expression was low, but integrin β ₃ was weakly positive in contrast to the BMDCs of the MHC class II-high population. gp49B expression was evident in the MHC class II-low population as well as in the MHC class II-high population. The MHC class II-low population is also negative for FN30 and FNIII _1-7 , indicating that the MHC class II-low population, like the MHC class II-high population, is tethered to the cell surface ( tethering) indicates that it does not have FN. Regarding splenic CD11c ⁺ DCs, integrin subunits α _V , β ₁ , and β ₃ were positive, but gp49, FN30, and FNIII expression were negative.

[Example 6] Examination of the correlation between gp49B and integrin β ₁ signals on BMDC Next, in the same manner as in Example 2, the fluorescence signals of gp49B and integrin β ₁ on the BMDC surface were measured using a confocal laser scanning microscope. Examined.
First, as a positive control, the signal correlation between MHC class I α chain and β ₂ microglobulin (β ₂ m) was analyzed. The results are shown in FIG. As shown in Figure 13, there is a positive linear correlation between the MHC class I α chain and β ₂ microglobulin (β ₂ m) signals (Pearson's correlation r = 0.594 ± 0.179, mean ± SD, n =6). Next, the fluorescent signals of gp49B and integrin _β1 were analyzed. As a result, although the fluorescence signal intensity was quite low, the fluorescence signal profile was entirely circular for both gp49 and integrin _β1 , as well as for MHC class Iα and _β2m .

Next, the correlation between the two fluorescent dyes was analyzed for the signals observed on the cell contours. The results are shown in FIG. As shown in Figure 13, the Pearson correlation between gp49 and integrin _β1 did not reach the preset significance level, but showed a weak correlation (r = 0.194 ± 0.071, mean ± SD, n=6), a relationship was observed between integrin and gp49B even in the absence of FN. This indicates that gp49B and some integrin molecules are located close to each other on the cell membrane. When FN is cross-linked between gp49B and integrin, such as by cell adhesion to a FN-coated dish, the proximity between gp49B and integrin becomes tighter.
From the above results, it is clear that gp49B and integrin _β1 on the BMDC surface are at least partially in close spatial proximity even in the absence of FN, which is a shared ligand between integrin and gp49B. Ta.

[Example 7] Tyrosine phosphorylation of Syk in BMDCs Next, we investigated whether binding of FN to integrins in BMDCs induces phosphorylation of FAK as a direct downstream signal of ligand-activated integrins. Specifically, the study was conducted using the following steps.
Wild-type BMDCs or gp49B-deficient BMDCs were seeded and cultured on FN-coated culture plates, respectively, and cell lysates were prepared for Western blot analysis. Next, the phosphorylation of FAK and Syk was examined in the obtained cell lysate. The results are shown in FIGS. 14A and 14B. As shown in FIG. 14A, culturing BMDC induced substantial tyrosine phosphorylation of FAK in wild-type BMDC. Substantial FAK phosphorylation comparable to that of wild-type BMDC was also induced in gp49B-deficient BMDC. This result suggested that FAK is not a direct target of potential gp49B-mediated regulation.

Next, we investigated whether Syk phosphorylation is regulated by gp49B in BMDCs seeded on immobilized FN. The results are shown in FIG. 14B. As shown in Figure 14B, phosphorylated Syk levels were increased upon FN-mediated stimulation of wild-type BMDCs, but the levels were further increased in gp49B-deficient BMDCs, suggesting that direct or Indirect gp49B-mediated suppression was suggested.
The above results revealed that immobilized FN directly induced downstream FAK activation, but this FN integrin-mediated signal was not affected by the presence or absence of gp49B. On the other hand, as shown in Figure 1, the pro-inflammatory effect of Syk via FN-integrin is due to the activation of Src family kinase (SFK) by immunoreceptor inhibitory tyrosine motif (ITIM) in BMDC. It has been revealed that this is suppressed by phosphorylated gp49B.

[Example 7] Relationship between LILRB4/gp49B and integrin in NK cells that do not have FN tethered to the cell surface CD3 ⁻ NK1.1 ⁺ CD49b ⁺ was detected from spleen cells of C57BL/6 mice using BD FACS Aria ^™ III. The cells were isolated as NK cells and cultured for 7 days in the presence of recombinant human IL-2 (800 U/ml). On the 7th day, 1 ng/ml of IL-12 was added, and 2 days after the addition, integrin β ₁ and gp49B on the NK cell surface were stained with fluorescently labeled antibodies, and confocal laser scanning was performed in the same manner as in Example 2. It was observed with a fluorescence microscope and the Pearson coefficient was calculated. The results are shown in FIG.
As shown in FIG. 15, the Pearson correlation coefficient between integrin β ₁ and gp49B was significantly higher in NK cells than in macrophages and dendritic cells (r=0.44, n=15). These results revealed that in NK cells, integrin _β1 and gp49B are located in a close position on the cell surface.

[Example 8] Suppressive effect of LILRB4/gp49 in microglial cells that do not have FN tethered to the cell surface Wild type (WT) and gp49-deficient mice (KO) of C57BL/6 mice 0-2 days old The cerebrum was removed, shredded and treated with trypsin, then seeded in a culture dish, and 14 days later the cells were collected by shaking. The expression of microglia-specific molecules CD11b and P2RY12 was confirmed for the collected cells by flow cytometry and immunohistochemistry.
The microglial cells obtained above were seeded on an FN-coated culture dish or a FN-uncoated culture dish, and the morphology of the cells was observed under a microscope after 24 hours. The results are shown in FIG. As shown in Figure 16, microglial cells isolated from wild-type mice and LILRB4 gene-deficient mice have different morphology when seeded, and microglial cells derived from wild-type mice have a ramified morphology. In contrast, microglial cells derived from LILRB4-deficient mice had a predominant form with more activated short processes (amoeboid). Next, 24 hours after seeding on FN-coated culture dishes or non-FN-coated culture dishes, the supernatant was collected, and the inflammatory cytokine TNF-α contained in the supernatant was measured by ELISA. The results are shown in FIG.
As shown in Figure 17, in microglial cells seeded on FN-coated culture dishes, secretion of TNF-α, which is one of the markers of inflammatory cytokines, was higher in microglial cells derived from LILRB4-deficient mice than in microglial cells derived from wild-type mice. It was higher in microglial cells. This result suggested that by inactivating LILRB4 in microglial cells, the suppression of the proinflammatory effect of microglial cells by LILRB4-FN is released.

Next, the long axis length of each microglial cell was measured based on the microscopic image. The results are shown in FIG. As shown in Figure 18, microglial cells isolated from wild-type mice and LILRB4 gene-deficient mice had different long axis lengths when seeded on FN-uncoated and FN-coated plates. It is clear that most of the cells are in the Ramified form (in resting phase), which spreads out for a long time, whereas the microglial cells isolated from LILRB4-deficient mice are mostly in the Amoeboid form (in the activated state), where they adhere without spreading. became. This suggests that LILRB4-deficient microglial cells are no longer suppressed by LILRB4-FN and are in a more activated state. As shown in Figure 18, the long axis length is longer when seeded on FN-coated plates than on non-FN-coated plates, so inactivation of microglial cells by LILRB4-FN is The involvement of FN-binding integrin was suggested.

Claims (17)

1. An activator for immune cells containing as an active ingredient a substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4.
2. The immune cell activator according to claim 1, wherein the activation of the immune cells is an inflammation-inducing effect of the immune cells.
3. The immune cell activator according to claim 2, wherein the immune cell inflammatory action is due to release of suppression by the immunosuppressive receptor LILRB4 against the immune cell inflammatory action based on integrin activation.
4. The immune cell activator according to claim 3, wherein the activation of the integrin is due to binding of fibronectin to the integrin.
5. The immune cell activator according to any one of claims 1 to 4, wherein the fibronectin binds to the immunosuppressive receptor LILRB4 via the amino acid sequence shown by SEQ ID NO: 1 in the fibronectin.
6. Any one of claims 1 to 4, wherein the substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4 is an anti-fibronectin antibody or a derivative thereof, an anti-immunosuppressive receptor LILRB4 antibody or a derivative thereof, or a fibronectin analog. The immune cell activator according to item 1.
7. The immune cell activating agent according to claim 6, wherein the anti-fibronectin antibody or its derivative binds to the amino acid sequence shown by SEQ ID NO: 1 in fibronectin.
8. The immune cell activating agent according to claim 6, wherein the fibronectin analog is a peptide of any one of the following (a) to (c).
   (a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1,
   (b) Contains an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and has a binding site for fibronectin of the immunosuppressive receptor LILRB4. a peptide with binding ability,
   (c) A peptide that includes an amino acid sequence having 80% or more identity in the amino acid sequence represented by SEQ ID NO: 1 and has the ability to bind to the fibronectin binding site of the immunosuppressive receptor LILRB4.
9. The agent for activating immune cells according to any one of claims 1 to 4, wherein the immune cells are selected from the group consisting of macrophages, microglial cells, dendritic cells, and NK cells.
10. A therapeutic agent for immune cell-related inflammatory diseases containing as an active ingredient a substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4.
11. The therapeutic agent according to claim 10, wherein the fibronectin binds to the immunosuppressive receptor LILRB4 via the amino acid sequence represented by SEQ ID NO: 1 in the fibronectin.
12. 12. The substance that inhibits the binding between fibronectin and the immunosuppressive receptor LILRB4 is an anti-fibronectin antibody or a derivative thereof, an anti-immunosuppressive receptor LILRB4 antibody or a derivative thereof, or a fibronectin analog. therapeutic agent.
13. The therapeutic agent according to claim 12, wherein the anti-fibronectin antibody or its derivative binds to the amino acid sequence represented by SEQ ID NO: 1 in fibronectin.
14. The therapeutic agent according to claim 12, wherein the fibronectin analog is any one of the following peptides (a) to (c).
    (a) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1,
    (b) Contains an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and has a binding site for fibronectin of the immunosuppressive receptor LILRB4. a peptide with binding ability,
    (c) A peptide that includes an amino acid sequence having 80% or more identity in the amino acid sequence represented by SEQ ID NO: 1 and has the ability to bind to the fibronectin binding site of the immunosuppressive receptor LILRB4.
15. The therapeutic agent according to claim 10 or 11, wherein the immune cells are selected from the group consisting of macrophages, microglial cells, dendritic cells, and NK cells.
16. 12. The immune cell-related inflammatory disease is selected from the group consisting of neurodegenerative diseases, mental developmental disorders, schizophrenia, autism spectrum disorders, atherosclerosis, and infectious diseases. therapeutic agent.
17. The therapeutic agent according to claim 16, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, dementia, cerebral infarction, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

Images