Immune checkpoint inhibitor and therapeutic agent for immune checkpoint-related disease

Abstract

Provided are an immune checkpoint inhibitor containing as an active ingredient a substance that inhibits the interaction of integrin and immunosuppressive receptor (referred to hereinafter as LILR) B3 and a therapeutic agent for immune checkpoint-related disease containing as an active ingredient a substance that inhibits the interaction of integrin and LILRB3.

Description

Immune checkpoint inhibitors and therapeutic agents for immune checkpoint-related diseases

[0001] The present invention relates to an immune checkpoint inhibitor and a therapeutic agent for an immune checkpoint-related disease.

[0002] The interaction between human immunosuppressive receptors and their physiological ligands is collectively referred to as immune checkpoints. Among these receptors, leukocyte Ig-like receptor (LILR) B is widely expressed on the surface of myeloid cells such as monocytes and macrophages. Members of the LILRB family known to date include LILRB1, LILRB2, LILRB3, LILRB4, and LILRB5. Among these, it has been reported that LILRB4 can determine inflammatory diseases resulting from immune system cell infection or autoimmunity (Patent Document 1). It has also been reported that the physiological ligand for LILRB4 is fibronectin, and that substances that inhibit the binding of LILRB4 to fibronectin are effective in treating immune checkpoint-related diseases (Patent Document 2).

[0003] LILRB1, LILRB2, and LILRB5 are known to be receptors for MHC class I molecules (Non-Patent Document 1), but there is insufficient evidence that they are functionally important and major physiological ligands. For LILRB3, several candidate ligand molecules, such as ApoE4, MHC class I, and Angptl, have been reported (Non-Patent Documents 2-4), but there is no consensus as to whether any of these molecules is a functionally important and major physiological ligand.

[0004] Various cells in the body maintain homeostasis while exerting functions such as activation, proliferation, migration, and so on as needed. To maintain this homeostasis and function, cells must form localized structures called focal adhesions with extracellular matrices composed of collagen, fibronectin, laminin, proteoglycans, and other molecules on the surfaces of cells and tissues in the surrounding environment, and then transduce signals into their own cells. The receptors primarily used to form these focal adhesions are a group of molecules collectively known as integrins (hereinafter also referred to as ITGs). It has been reported that the most upstream signal transduced into cells is the activation of two types of tyrosine kinases: activation by phosphorylation of focal adhesion kinase (FAK), which induces the reorganization of actin filaments, and activation by phosphorylation of spleen tyrosine kinase (Syk), which has the ability to induce inflammation (Non-Patent Document 5).

[0005] ITG is a heterodimer consisting of two subunits, an α-chain and a β-chain, and there are many different α-chains and β-chains, with a wide variety of combinations. ITG is an essential molecule for cell adhesion, cell migration, cell proliferation, and other phenomena that require actin reorganization and polymerization. It is known to be involved in various cellular phenomena, including various immune responses and tissue repair to maintain normal life activities, as well as inflammatory responses, thrombus formation, tumor formation / metastasis, organogenesis, and maintenance of organ function (Non-Patent Documents 6-8). ITG also promotes cell-cell interactions, cell adhesion to vascular walls, cell migration, and interactions with complement fragments. Genetic deficiencies in ITG are known to cause infectious diseases due to leukocyte adhesion deficiency and bacterial phagocytosis deficiency (Non-Patent Document 9), and to contribute to neuronal process outgrowth and the formation of circuits between neurons (Non-Patent Document 10).

[0006] Regarding the relationship between ITG and LILRB, it has been reported that the activation of ITG, which occurs when fibronectin binds to ITG on the cell surface, is inhibited by the binding of fibronectin to LILRB4 on the cell surface, and that substances that inhibit the binding of fibronectin to LILRB4 can release the inhibition of ITG activation by fibronectin caused by LILRB4, thereby activating ITG (Patent Document 3, Non-Patent Document 11).

[0007] JP 2018-25554 A International Publication No. 2021 / 029318 International Publication No. 2023 / 233791

[0008] J Immunol (2016) 196 (3): 947-955.Cell Res. 2023 Feb;33(2):116-130.Nat Immunol. 2021 Nov;22(11):1391-1402.Nature 2012, 485: 656-660.Cancer Immunol. Res., 2021, 9(11):1283-1297.Pediatric Res., 2021, 89, 1619-1626.Thrombosis and Hemostasis, 2013, 24(5):507-515.Protein Nucleic Acid Enzyme, 1999, 44(2):130-135.MSD Manual Professional Edition (https: / / www.msdmanuals.com / ja-jp / professional) 12. Immunology "Leukocyte adhesion deficiency" Japanese Journal of Pharmacology, 2018, 152: 240-245. International Immunology, 2022, 34(8):435-444.

[0009] An object of the present invention is to provide an immune checkpoint inhibitor and a therapeutic agent for immune checkpoint-related diseases.

[0010] The present inventors have found that LILRB3 interacts with the ITGβ subunit and that a substance that inhibits the interaction between LILRB3 and ITG is useful as an immune checkpoint inhibitor and a therapeutic agent for immune checkpoint-related diseases. Furthermore, the present inventors have found that LILRB directly binds to the ITGβ subunit and that a substance that inhibits the binding of LILRB to ITG is useful as an immune checkpoint inhibitor and a therapeutic agent for immune checkpoint-related diseases. Based on these findings, the present invention has been completed. A first embodiment of the present invention includes the following aspects: [1] An immune checkpoint inhibitor comprising, as an active ingredient, a substance that inhibits the interaction between ITG and LILRB3. [2] The immune checkpoint inhibitor according to [1], wherein the interaction between ITG and LILRB3 includes the interaction between the ITGβ1 subunit and LILRB3. [3] The immune checkpoint inhibitor according to [1] or [2], wherein the substance that inhibits the interaction between ITG and LILRB3 is an anti-LILRB3 antibody or a derivative thereof. [4] A therapeutic agent for immune checkpoint-related diseases, comprising as an active ingredient a substance that inhibits the interaction between ITG and LILRB3. [5] The therapeutic agent for immune checkpoint-related diseases according to [4], wherein the immune checkpoint-related disease is selected from the group consisting of autoimmune diseases, cancer, inflammatory diseases, Alzheimer's disease, infectious diseases, and allergic diseases. [6] The therapeutic agent for immune checkpoint-related diseases according to [4] or [5], wherein the interaction between ITG and LILRB3 includes the interaction between the ITGβ1 subunit and LILRB3. [7] The therapeutic agent for immune checkpoint-related diseases according to [4] or [5], wherein the substance that inhibits the interaction between ITG and LILRB3 is an anti-LILRB3 antibody or a derivative thereof.

[0011] The second embodiment of the present invention also includes the following aspects. [8] An immune checkpoint inhibitor comprising, as an active ingredient, a substance that inhibits the binding between ITG and LILRB. [9] The immune checkpoint inhibitor according to [8], wherein the LILRB is LILRB2.

[10] The immune checkpoint inhibitor according to [9], wherein the binding between ITG and LILRB2 comprises the binding between the ITGβ1 subunit and LILRB2.

[11] The immune checkpoint inhibitor according to [9] or

[10] , wherein the substance that inhibits the binding between ITG and LILRB2 is an anti-LILRB2 antibody or a derivative thereof.

[12] The immune checkpoint inhibitor according to [8], wherein the LILRB is LILRB5.

[13] The immune checkpoint inhibitor according to

[12] , wherein the binding between ITG and LILRB5 comprises the binding between LILRB5 and at least one ITG subunit selected from the group consisting of the ITGβ1 subunit and the ITGβ3 subunit.

[14] The immune checkpoint inhibitor according to

[12] or

[13] , wherein the substance that inhibits the binding between ITG and LILRB5 is an anti-LILRB5 antibody or a derivative thereof.

[15] A therapeutic agent for immune checkpoint-related diseases, comprising as an active ingredient a substance that inhibits the binding between ITG and LILRB.

[16] The therapeutic agent for immune checkpoint-related diseases according to

[15] , wherein the immune checkpoint-related disease is selected from the group consisting of autoimmune diseases, cancer, inflammatory diseases, Alzheimer's disease, infectious diseases, and allergic diseases.

[17] The therapeutic agent for immune checkpoint-related diseases according to

[15] or

[16] , wherein the LILRB is LILRB2.

[18] The therapeutic agent for immune checkpoint-related diseases according to

[17] , wherein the binding between ITG and LILRB2 includes the binding between the ITGβ1 subunit and LILRB2.

[19] The therapeutic agent for immune checkpoint-associated diseases according to

[17] or

[18] , wherein the substance that inhibits the binding of ITG and LILRB2 is an anti-LILRB2 antibody or a derivative thereof.

[20] The therapeutic agent for immune checkpoint-associated diseases according to

[15] or

[16] , wherein the LILRB is LILRB5.

[21] The therapeutic agent for immune checkpoint-associated diseases according to

[20] , wherein the binding between ITG and LILRB5 comprises binding between LILRB5 and at least one ITG subunit selected from the group consisting of ITGβ1 subunit and ITGβ3 subunit.

[22] The therapeutic agent for immune checkpoint-associated diseases according to

[20] or

[21] , wherein the substance that inhibits the binding between ITG and LILRB5 is an anti-LILRB5 antibody or a derivative thereof.

[0012] According to the present invention, it is possible to provide an immune checkpoint inhibitor and a therapeutic agent for an immune checkpoint-related disease.

[0013] Fig. 1 shows the results of biolayer interferometry (BLI) analysis in Example 1. Fig. 2 shows the affinity of LILRB2 and LILRB5 for the ITGβ1 subunit. ＋ FIG. 1 shows the results of flow cytometry analysis of the expression of LILRB isoforms on the surface of monocytes. ＋ FIG. 1 shows the results of flow cytometry analysis of the expression of ITG subunits on the surface of monocytes. ＋

[0039] Figure 1 shows the results of flow cytometry analysis of the expression of fibronectin (FN) and the N-terminal 30 kDa domain of fibronectin (FN30) on the surface of monocytes. ＋

[0039] Figure 1 shows images of the surface of monocytes captured by a confocal laser scanning microscope. CD14 double-stained with a monoclonal antibody against LILRB2, LILRB3, or LILRB4 and an antibody against the ITG β1 subunit. ＋This figure shows the results of calculating Pearson's correlation coefficient r from each signal profile of monocyte cell contours. The upper graph shows the Pearson's correlation coefficient r for each LILRB isoform in FN-uncoated or FN-coated dishes, where NS indicates no significant difference. The lower graph shows the Pearson's correlation coefficient r for each LILRB isoform in FN-coated or non-coated dishes. CD14 stained with anti-LILRB3 monoclonal antibody or anti-ITGβ1 subunit antibody ＋1 shows images of monocytes captured with a confocal laser scanning microscope. The top row shows images stained with the above antibodies individually, the middle row shows images stained with the above antibodies simultaneously, and the bottom row shows images stained first with an anti-ITGβ1 subunit antibody and then with an anti-LILRB3 monoclonal antibody. The figures show the results of flow cytometry analysis of cells stained with a secondary antibody against the anti-ITGβ1 antibody after simultaneously adding the anti-LILRB3 monoclonal antibodies #222821 or #08, or their corresponding isotype antibodies, when adding the anti-ITGβ1 subunit antibody. The isotype antibody in the figure indicates the isotype antibody corresponding to the anti-ITGβ1 antibody. The figures show the results of flow cytometry analysis of cells to which the anti-LILRB3 monoclonal antibodies #222821 or #08 and the anti-ITGβ1 subunit antibody were simultaneously added, stained with a secondary antibody against the anti-ITGβ1 antibody, and then analyzed. "Isotype" in the figure indicates that the fluorescence intensity of the isotype antibody corresponding to the anti-ITG antibody and the anti-LILRB3 antibody was set to 100%. This figure shows the interference of anti-LILRB3 monoclonal antibody with each of the ITG subunit antibodies α4, α5, and αM, measured by flow cytometry. The interference of anti-LILRB3 monoclonal antibody with each of the ITG subunit antibodies α5 and β2 was measured by flow cytometry, and the interference of anti-LILRB2 antibody with anti-ITG α5 antibody was statistically analyzed, and the interference of anti-LILRB2 antibody with anti-ITG β2 antibody was measured. This figure shows the interference of anti-LILRB2 monoclonal antibody with each of the ITG subunit antibodies β1, β2, β3, α4, α5, and αM, measured by flow cytometry.

[0033] Figure 1 shows the results of flow cytometry measurement and statistical analysis of the interference of anti-LILRB2 monoclonal antibody with ITG β1, α4, and αM subunit antibodies. Fluorescence images are shown measured by fluorescence resonance energy transfer (FRET) using an anti-ITG β1 subunit antibody as the donor and an anti-LILRB3 antibody or isotype antibody as the acceptor. The upper row shows the fluorescence image before bleaching, and the lower row shows the fluorescence image after bleaching.

[0033] Figure 1 shows the results of calculating FRET efficiency from fluorescence images measured by the fluorescence resonance energy transfer (FRET) method using an anti-ITGβ1 subunit antibody as a donor and an anti-LILRB3 antibody or an isotype antibody as an acceptor on an FN-coated or non-coated dish. Figure 1 shows fluorescence images measured by the fluorescence resonance energy transfer (FRET) method using an anti-ITGβ1 subunit antibody as a donor and an anti-LILRB2 antibody or an isotype antibody as an acceptor. The upper row shows fluorescence images before bleaching, and the lower row shows fluorescence images after bleaching.

[0034] Figure 1 shows the results of calculating FRET efficiency from fluorescence images measured by the fluorescence resonance energy transfer (FRET) method using an anti-ITGβ1 subunit antibody as a donor and an anti-LILRB2 antibody or an isotype antibody as an acceptor on an FN-coated or non-coated dish. 1 shows the results of Western blotting analysis of FAK and Syk phosphorylation using anti-LILRB3 monoclonal antibody #08, anti-LILRB2 monoclonal antibody 42D1, anti-LILRB4 monoclonal antibody ZM4.1, or a combination of these antibodies.

[0034] FIG. 1 shows the results of statistical analysis of the results of Western blotting analysis of FAK and Syk phosphorylation using anti-LILRB3 monoclonal antibody #08, anti-LILRB2 monoclonal antibody 42D1, anti-LILRB4 monoclonal antibody ZM4.1, or a combination of these antibodies. The upper row shows the results for FAK phosphorylation, and the lower row shows the results for Syk phosphorylation.

[0035] FIG. 1 shows changes in cell morphology of monocytes treated with anti-LILRB2 monoclonal antibody or anti-LILRB3 monoclonal antibody. The upper row shows microscopic photographs, and the lower row shows the longitudinal length of the cells. Figures showing the release of TNF-α or IL-6 from monocytes treated with anti-LILRB2 monoclonal antibody or anti-LILRB3 monoclonal antibody. The upper figure shows the release of TNF-α, and the lower figure shows the release of IL-6. Figures showing the results of BLI analysis in Example 9. PIR-B and integrin β2 expressed in the mouse macrophage cell line RAW264.7 were stained with fluorescently labeled antibodies, respectively, and images were captured using a confocal laser fluorescence microscope, followed by the Pearson's correlation coefficient r measured for the fluorescent signals at the focal plane of cell adhesion.1 shows the competitive action between anti-PIR-A / B monoclonal antibodies and anti-ITG β1 subunit antibodies in the mouse macrophage cell line RAW264.7. The left graph shows the results when anti-PIR-A / B D1D2 monoclonal antibody 6C1 or anti-PIR-A / B D5D6 monoclonal antibody 11.3 was used in combination with an anti-ITG β1 subunit antibody, and the right graph shows the results when anti-PIR-A / B D5D6 monoclonal antibody 10.1 was used in combination with an anti-ITG β1 subunit antibody. 1 is a graph showing a statistical analysis of interference between anti-PIR-A / B D5D6 monoclonal antibody 11.3 and anti-ITGβ1 subunit antibody in the mouse macrophage cell line RAW264.7; and FIG. 2 is a graph showing a statistical analysis of interference between anti-PIR-A / B monoclonal antibody and anti-ITGβ2 subunit monoclonal antibody in the mouse macrophage cell line RAW264.7. This figure shows the results of Western blotting analysis of FAK and Syk phosphorylation using anti-PIR-A / B monoclonal antibody 11.3 in mouse peritoneal cells. This figure shows a statistical analysis of the results of Western blotting analysis of FAK and Syk phosphorylation using anti-PIR-A / B monoclonal antibody 11.3 in mouse peritoneal cells. The left figure shows the results for FAK phosphorylation, and the right figure shows the results for Syk phosphorylation.

[0014] [Immune Checkpoint Inhibitor] The immune checkpoint inhibitor according to the first embodiment of the present invention contains, as an active ingredient, a substance that inhibits the interaction between ITG and LILRB3. In this embodiment, the substance that inhibits the interaction between ITG and LILRB3 is not particularly limited as long as it has the activity of inhibiting the interaction between ITG and LILRB3, and examples thereof include an anti-LILRB3 antibody or a derivative thereof.

[0015] The anti-LILRB3 antibody may be either a monoclonal or polyclonal antibody as long as it reacts with LILRB3, with monoclonal antibodies being preferred. Such antibodies can be produced by well-known methods. For example, to produce polyclonal antibodies, mice, rats, hamsters, rabbits, goats, sheep, chickens, and the like are used as immunized animals. Antisera can be obtained from serum after administering an antigen once or multiple times to an animal subcutaneously, intradermally, intraperitoneally, or the like. When a protein or peptide is used as the antigen, immunization with a mixture of the antigen and a replacement fluid having an immunostimulatory effect is more preferred.

[0016] Monoclonal antibodies can be produced according to known monoclonal antibody production methods, such as those described in "Monoclonal Antibodies" by Kaoru Nagamune and Hiroshi Terada, Hirokawa Shoten (1990), or James W. Golding, "Monoclonal Antibody", 3rd edition, Academic Press, 1996. Monoclonal antibodies can also be produced by DNA immunization, and can be produced with reference to Nature 1992 Mar12;356 152-154 and J. Immunol Methods Mar1;249 147-154.

[0017] The anti-LILRB3 monoclonal antibody can be produced by culturing a hybridoma prepared according to a conventional method and isolating it from the culture supernatant, or by administering the hybridoma to a mammalian animal compatible with the hybridoma and recovering it as ascites. The anti-LILRB3 monoclonal antibody can also be produced using known genetic recombination techniques. Specifically, the monoclonal antibody produced by the hybridoma prepared above and a gene encoding the antibody are cloned to prepare a vector containing the gene, which is then introduced into host cells for transformation to obtain cells expressing the anti-LILRB3 antibody, and the resulting cells are then cultured. The cells, vector type, cell type, culture conditions, and other factors used in this preparation are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.

[0018] The antibody can be further purified before use, if necessary. Techniques for purifying and isolating the antibody include conventionally known methods, such as salting out (e.g., ammonium sulfate precipitation), gel filtration (e.g., using Sephadex), ion exchange chromatography, and affinity purification (e.g., using a protein A column).

[0019] The anti-LILRB3 antibody may be either a monoclonal antibody or a polyclonal antibody as long as it binds to LILRB3, but a monoclonal antibody is preferably used.

[0020] The antigen used to produce anti-LILRB3 antibodies can be the LILRB3 protein, a fragment thereof (peptide), or a vector incorporating cDNA encoding the LILRB3 protein. To obtain monoclonal antibodies that recognize the higher-order structure of LILRB3, a full-length LILRB3 vector containing the full-length human LILRB3 gene is the optimal immunization antigen gene. However, gene constructs into which a partial region of the LILRB3 sequence has been inserted can also be used as immunization antigen genes. DNA immunization can be performed by subcutaneously injecting the above gene constructs, either alone or in combination, into an animal (such as a mouse or rat) using any of a variety of gene transfer methods (e.g., intramuscular injection, electroporation, gene gun, etc.) and allowing them to be incorporated into the cells.

[0021] The derivative of the anti-LILRB3 antibody includes, for example, F(ab') of the anti-LILRB3 antibody. ２ , F(ab) ２ , Fab', Fab, Fv, scFv, variants thereof, fusion proteins or fusion peptides containing an antibody portion, and the like.

[0022] In the immune checkpoint inhibitors of the present invention, the anti-LILRB3 antibody or derivative thereof can inhibit the interaction between ITG and LILRB3. In the present invention, the interaction between ITG and LILRB3 refers to the action of LILRB3 and ITG on ITG through a direct binding that is weak enough to be undetectable by BLI analysis, or to the indirect action, for example, the action of LILRB3 on ITG directly or indirectly due to their spatially close or adjacent relationship. The interaction of the present invention also includes the action of LILRB3 on ITG via some molecule.

[0023] The interaction between ITG and LILRB3 may be an interaction between any ITG subunit and LILRB3 as long as it is an interaction between ITG and LILRB3, but an interaction between LILRB3 and the ITGβ1 subunit is preferred, and may be an interaction with a single ITG subunit or an interaction with multiple ITG subunits.

[0024] Inhibition of the interaction between ITG and LILRB3 can be achieved by evaluating the inhibition of the interaction between ITG and cells expressing LILRB3. The cells expressing LILRB3 are not particularly limited as long as they express LILRB3, and examples thereof include peripheral blood leukocytes, peripheral blood mononuclear cells, spleen cells, bone marrow cells, brain cells, and monocytes / macrophages isolated therefrom, dendritic cells, eosinophils, basophils, neutrophils, mast cells, activated T cells, B cells, plasma cells, and microglial cells.

[0025] The nucleotide sequence and amino acid sequence of LILRB3 can be found in the database provided by the National Center for Biotechnology Information (NCBI). For human (Homo sapiens) LILRB3, for example, the Entrez GeneID is 11025 (as of November 23, 2023), and the RefSeq ProteinIDs are NP_001074919.2, NP_001307889.1, and NP_006855.3 (corresponding to isoforms 1 to 3). Examples of mouse (Mus musculus) LILRB3 include Gene ID 18733 (as of November 23, 2023) and RefSeq Protein IDs NP_001344323.1 and NP_035225.2 (corresponding to isoforms 1 and 2), and examples of rat (Rattus norvegicus) LILRB3 include Gene ID 308350 (as of November 23, 2023) and RefSeq Protein ID XP_038956583.1, and other animals are known to have LILRB3. The LILRB3 of the present invention is not limited to the above LILRB3s, and other LILRB3s are also included in the LILRB3 of the present invention.

[0026] In cells expressing ITG on the cell surface, LILRB3 interacts with an ITG subunit, preferably the β1 subunit of ITG, thereby suppressing ITG-mediated activation of the cells. When LILRB3 interacts with ITG, phosphorylation of spleen tyrosine kinase (hereinafter referred to as "Syk") downstream of ITG is suppressed. Inhibition of Syk phosphorylation suppresses inflammation-inducing actions such as the secretion of inflammatory cytokines, cell activation, cell proliferation, cell differentiation, phagocytosis, and the like.

[0027] A substance that inhibits the interaction between ITG and LILRB3 inhibits the interaction between ITG and LILRB3, thereby inducing the phosphorylation of Syk that is suppressed by the interaction of LILRB3 with ITG, and enhancing pro-inflammatory effects, etc.

[0028] In this embodiment, the ITG is not particularly limited as long as it is expressed on the cell surface. Examples of cells expressing ITG on their surface include myeloid cells such as macrophages, microglial cells, and dendritic cells; lymphoid cells such as NK cells, T cells, and B cells; immune cells such as monocytes, granulocytes, mast cells, and basophils; platelets; epithelial cells; skeletal muscle cells; and nerve cells, with monocytes / macrophages, neutrophils, microglial cells, dendritic cells, T cells, B cells, and NK cells being preferred.

[0029] The immune checkpoint inhibitor of this embodiment contains, as an active ingredient, a substance that inhibits the interaction between ITG and LILRB3, and may further contain pharmaceutically acceptable carriers and additives.

[0030] Examples of carriers and additives include, but are not limited to, water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, 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.

[0031] The immune checkpoint inhibitor of this embodiment can be in various forms, such as a liquid (e.g., an injection), a dispersion, a suspension, a tablet, a pill, a powder, a suppository, etc. A preferred embodiment is an injection, which is preferably administered parenterally (e.g., intravenously, transdermally, intraperitoneally, intramuscularly).

[0032] The immune checkpoint inhibitor of this embodiment can be used as a therapeutic agent for immune checkpoint-associated diseases. The dose of the immune checkpoint inhibitor of this embodiment can be, for example, but is not limited to, 0.025 to 50 mg / kg, preferably 0.1 to 50 mg / kg, more preferably 0.1 to 25 mg / kg, and even more preferably 0.1 to 10 mg / kg or 0.1 to 3 mg / kg.

[0033] An immune checkpoint inhibitor according to a second embodiment of the present invention comprises, as an active ingredient, a substance that inhibits the binding of ITG and LILRB. In this embodiment, the LILRB is not particularly limited, but LILRB2 and LILRB5 are preferred. The substance that inhibits the binding of ITG and LILRB is not particularly limited as long as it has the activity of inhibiting the binding of ITG and LILRB, and examples thereof include an anti-LILRB antibody or a derivative thereof. For example, an example of a substance that inhibits the binding of ITG and LILRB2 is an anti-LILRB2 antibody or a derivative thereof, and an example of a substance that inhibits the binding of ITG and LILRB5 is an anti-LILRB5 antibody or a derivative thereof.

[0034] The anti-LILRB antibody may be either a monoclonal or polyclonal antibody as long as it reacts with LILRB, but a monoclonal antibody is preferably used. The antibody can be produced by the same method as the LILRB3 antibody in the first embodiment.

[0035] The derivative of the anti-LILRB2 antibody includes, for example, F(ab') of the anti-LILRB2 antibody. ２ , F(ab) ２ , Fab', Fab, Fv, scFv, variants thereof, fusion proteins or fusion peptides containing an antibody portion, and the like.

[0036] The derivative of the anti-LILRB5 antibody includes, for example, F(ab') of the anti-LILRB5 antibody. ２ , F(ab) ２ , Fab', Fab, Fv, scFv, variants thereof, fusion proteins or fusion peptides containing an antibody portion, and the like.

[0037] The binding between ITG and LILRB2 may be an interaction between any subunit of ITG and LILRB2, provided that it is a binding between ITG and LILRB2, but the binding between LILRB2 and the ITG β1 subunit is preferred.

[0038] The binding between ITG and LILRB5 may be an interaction between any subunit of ITG and LILRB5, as long as it is a binding between ITG and LILRB5, but the binding between LILRB5 and the ITG β1 subunit or ITG β3 subunit is preferred.

[0039] Inhibition of the binding of ITG to LILRB2 can be achieved by evaluating inhibition of the binding of ITG to cells expressing LILRB2. The cells expressing LILRB2 are not particularly limited as long as they express LILRB2, and examples include spleen cells, peripheral blood mononuclear 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, and microglial cells.

[0040] Inhibition of the binding of ITG to LILRB5 can be achieved by evaluating inhibition of the binding of ITG to cells expressing LILRB5. The cells expressing LILRB5 are not particularly limited as long as they express LILRB5, and examples include 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, and microglial cells.

[0041] The nucleotide sequence and amino acid sequence of LILRB2 can be found in the database provided by the National Center for Biotechnology Information (NCBI). For human (Homo sapiens) LILRB2, for example, the Entrez GeneID is 10288 (as of November 23, 2023), and the RefSeq ProteinIDs are NP_001074447.2, NP_001265332.2, NP_001265333.2, NP_001265334.2, NP_001265335.2, and NP_005865.3 (corresponding to isoforms 1 to 5). Although no counterpart of mouse (Mus musculus) LILRB2 has been identified, examples of rat (Rattus norvegicus) LILRB2 include Gene ID 65146 (as of November 23, 2023) and RefSeq Protein ID NP_113901.2, and other animals are known to have LILRB2. The present invention is not limited to the above LILRB2, and other LILRB2s are also included in the LILRB2 of the present invention.

[0042] In cells expressing ITG on the cell surface, LILRB2 binds to an ITG subunit, preferably the β1 subunit of ITG, thereby suppressing activation of ITG in the cells. When LILRB2 binds to ITG, phosphorylation of Syk downstream of ITG is suppressed. Inhibition of Syk phosphorylation suppresses inflammation-inducing actions such as the secretion of inflammatory cytokines, cell activation, cell proliferation, cell differentiation, phagocytosis, and the like.

[0043] A substance that inhibits the binding of ITG to LILRB2 inhibits the binding of ITG to LILRB2, thereby inducing the phosphorylation of Syk that is suppressed by the binding of LILRB2 to ITG, and enhancing pro-inflammatory effects, etc.

[0044] The nucleotide sequence and amino acid sequence of LILRB5 can be found in the database provided by the National Center for Biotechnology Information (NCBI). For human (Homo sapiens) LILRB5, for example, the Entrez GeneID is 10990 (as of November 23, 2023), and the RefSeq ProteinIDs are NP_001074911.2, NP_001074912.2, NP_001291386.2, NP_001399198.1, and NP_006831.2 (corresponding to isoforms 1 to 5). While mouse (Mus musculus) LILRB5 and rat (Rattus norvegicus) LILRB5 have not been identified, other animals are known to have LILRB5. The present invention is not limited to the above LILRB5, and other LILRB5s are also included in the LILRB5 of the present invention.

[0045] In cells expressing ITG on the cell surface, LILRB5 binds to an ITG subunit, preferably the ITG β1 subunit or the ITG β3 subunit, thereby suppressing ITG-mediated activation of the cells. When LILRB5 binds to ITG, phosphorylation of Syk downstream of ITG is suppressed. Inhibition of Syk phosphorylation suppresses inflammation-inducing actions such as the secretion of inflammatory cytokines, cell activation, cell proliferation, cell differentiation, phagocytosis, and the like.

[0046] A substance that inhibits the binding of ITG to LILRB5 inhibits the binding of ITG to LILRB5, thereby inducing the phosphorylation of Syk, which is suppressed by the binding of LILRB5 to ITG, and enhancing pro-inflammatory effects, etc.

[0047] In this embodiment, the ITG is not particularly limited as long as it is expressed on the cell surface. Examples of cells expressing ITG on their surface include myeloid cells such as macrophages, microglial cells, and dendritic cells; lymphoid cells such as NK cells, T cells, and B cells; immune cells such as monocytes, granulocytes, mast cells, and basophils; platelets; epithelial cells; skeletal muscle cells; and nerve cells, with macrophages, microglial cells, dendritic cells, T cells, B cells, and NK cells being preferred.

[0048] The immune checkpoint inhibitor of this embodiment contains, as an active ingredient, a substance that inhibits the binding between ITG and LILRB2, and may further contain pharmaceutically acceptable carriers and additives.

[0049] Furthermore, the immune checkpoint inhibitor of this embodiment contains, as an active ingredient, a substance that inhibits the binding between ITG and LILRB5, and may further contain pharmaceutically acceptable carriers and additives.

[0050] In the second embodiment, the pharmaceutically acceptable carriers and additives can be the same as those in the first embodiment.

[0051] The immune checkpoint inhibitor of this embodiment can be in various forms, such as a liquid (e.g., an injection), a dispersion, a suspension, a tablet, a pill, a powder, a suppository, etc. A preferred embodiment is an injection, which is preferably administered parenterally (e.g., intravenously, transdermally, intraperitoneally, intramuscularly).

[0052] The immune checkpoint inhibitor of this embodiment can be used as a therapeutic agent for immune checkpoint-associated diseases. The dose of the immune checkpoint inhibitor of this embodiment can be, for example, but is not limited to, 0.025 to 50 mg / kg, preferably 0.1 to 50 mg / kg, more preferably 0.1 to 25 mg / kg, and even more preferably 0.1 to 10 mg / kg or 0.1 to 3 mg / kg.

[0053] [Therapeutic Agent for Immune Checkpoint-Associated Diseases] The therapeutic agent for immune checkpoint-associated diseases of the present invention contains, as an active ingredient, a substance that inhibits the interaction between ITG and LILRB3. The substance that inhibits the interaction between ITG and LILRB3 is not particularly limited as long as it has the activity of inhibiting the interaction between ITG and LILRB3, and examples include an anti-LILRB3 antibody or a derivative thereof. Examples of the anti-LILRB3 antibody or a derivative thereof include those described above.

[0054] Furthermore, the therapeutic agent for immune checkpoint-associated diseases of the present invention contains, as an active ingredient, a substance that inhibits the binding of ITG and LILRB. The substance that inhibits the binding of ITG and LILRB is not particularly limited as long as it has the activity of inhibiting the binding of ITG and LILRB, and examples thereof include an anti-LILRB2 antibody or a derivative thereof, an anti-LILRB5 antibody or a derivative thereof, etc. Examples of the anti-LILRB2 antibody or a derivative thereof and the anti-LILRB5 antibody or a derivative thereof include those described above.

[0055] In the present invention, the immune checkpoint-associated disease is not particularly limited as long as it is a disease in which LILRB3, an immune checkpoint molecule, is involved, and examples include autoimmune diseases, cancer, inflammatory diseases, Alzheimer's disease, infectious diseases, and allergic diseases.

[0056] Examples of autoimmune diseases include Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, vasculitis, Addison's disease, polymyositis, dermatomyositis, psoriasis, Sjögren's syndrome, systemic sclerosis, glomerulonephritis, etc. Examples of cancers include lung cancer, colon cancer, kidney cancer, malignant melanoma, Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, liver cancer, prostate cancer, osteosarcoma, leukemia, etc. Cancers may be primary or metastatic, but the present invention is preferably used for metastatic cancers.

[0057] Examples of inflammatory diseases include systemic lupus erythematosus, dermatomyositis, Kawasaki disease, psoriasis, herpes zoster, chronic obstructive pulmonary disease (COPD), bronchial asthma, atopic dermatitis, rheumatoid arthritis, antiphospholipid syndrome, polymyositis, vasculitis syndrome, Sjogren's syndrome, Behcet's disease, Graves' disease, Hashimoto's disease, myocarditis, aortitis syndrome, ulcerative colitis, Crohn's disease, primary biliary cirrhosis, autoimmune hepatitis, autoimmune pancreatitis, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, glomerulonephritis, ANCA-associated nephritis, amyloidosis, TINU syndrome, hypersensitivity pneumonitis, eosinophilic pneumonia, and sarcoidosis.

[0058] Examples of infectious diseases include influenza, Empox (monkeypox), hepatitis, infectious gastroenteritis, tuberculosis, diphtheria, invasive meningococcal infection, chickenpox, COVID-19 infection, hand, foot, and mouth disease, Japanese encephalitis, norovirus infection, pneumococcal infection, tetanus, human papillomavirus infection, pertussis, rubella, polio (acute poliomyelitis), measles, Legionnaires' disease, rotavirus infection, Hib infection, HIV / AIDS, and HTLV-1 (human T-cell leukemia virus type 1).

[0059] Examples of allergic diseases include allergic rhinitis, bronchial asthma, urticaria / atopic dermatitis, shingles, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergy, anaphylaxis, autoimmune hemolytic anemia, thrombocytopenia, granulocytopenia, neonatal hemolytic jaundice, serum sickness, hypersensitivity pneumonitis, lupus nephritis (chronic glomerulonephritis), systemic lupus erythematosus, contact dermatitis, Hashimoto's disease, Behcet's disease, rejection after organ transplantation, and graft-versus-host disease (GVHD).

[0060] When LILRB3 interacts with ITG, phosphorylation of Syk downstream of ITG is suppressed, which in turn suppresses pro-inflammatory actions such as the secretion of inflammatory cytokines.

[0061] A substance that inhibits the interaction between ITG and LILRB3 inhibits the interaction of ITG with LILRB3, thereby inducing the phosphorylation of Syk that is suppressed by the interaction of LILRB3 with ITG, thereby enhancing pro-inflammatory effects, etc. Therefore, a substance that inhibits the interaction between ITG and LILRB3 can treat immune checkpoint-related diseases that develop due to the inhibition of ITG activation.

[0062] Furthermore, when LILRB2 or LILRB5 binds to ITG, phosphorylation of Syk downstream of ITG is suppressed. When Syk phosphorylation is suppressed, pro-inflammatory actions such as the secretion of inflammatory cytokines are suppressed.

[0063] A substance that inhibits the binding of ITG to LILRB2 inhibits the binding of ITG to LILRB2, thereby inducing the phosphorylation of Syk, which is suppressed by the binding of LILRB2 to ITG, and enhancing pro-inflammatory effects, etc. Therefore, a substance that inhibits the binding of ITG to LILRB2 can treat immune checkpoint-related diseases that develop due to the inhibition of ITG activation.

[0064] Furthermore, a substance that inhibits the binding of ITG to LILRB5 inhibits the binding of ITG to LILRB5, thereby inducing the phosphorylation of Syk, which is suppressed by the binding of LILRB5 to ITG, and enhancing pro-inflammatory effects, etc. Therefore, a substance that inhibits the binding of ITG to LILRB5 can treat immune checkpoint-related diseases that develop due to the inhibition of ITG activation.

[0065] In the therapeutic agent for immune checkpoint-related diseases of the present invention, the ITG is not particularly limited as long as it is expressed on the cell surface. Examples of cells expressing ITG on their surface include myeloid cells such as monocytes / macrophages, neutrophils, microglial cells, and dendritic cells; lymphoid cells such as NK cells, T cells, and B cells; immune cells such as monocytes, granulocytes, mast cells, and basophils; platelets; epithelial cells; skeletal muscle cells; and nerve cells, with monocytes, macrophages, neutrophils, microglial cells, dendritic cells, T cells, B cells, and NK cells being preferred.

[0066] The therapeutic agent for immune checkpoint-associated diseases of the present invention may further contain pharmaceutically acceptable carriers and additives, such as those described above.

[0067] The therapeutic agent for immune checkpoint-associated diseases of the present invention can be in various forms, such as a liquid (e.g., an injection), a dispersion, a suspension, a tablet, a pill, a powder, a suppository, etc. A preferred embodiment is an injection, which is preferably administered parenterally (e.g., intravenously, transdermally, intraperitoneally, intramuscularly).

[0068] The dosage of the therapeutic agent for immune checkpoint-associated diseases of the present invention can be, for example, 0.025 to 50 mg / kg, preferably 0.1 to 50 mg / kg, more preferably 0.1 to 25 mg / kg, and even more preferably 0.1 to 10 mg / kg or 0.1 to 3 mg / kg, but is not limited to this.

[0069] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0070] Example 1 Biolayer Interferometry (BLI) Analysis In this example, clone GHI / 75 (BD Bioscience) was used as the anti-LILRB1 antibody, and clone 42D1 (BioLegend) was used as the anti-LILRB2 antibody. Clone MKT5.1 (BioLegend), clone #222821 (R&D Systems), and clone #08 (Sino Biological) were used as the anti-LILRB3 antibodies. Clone ZM4.1 (Invitrogen) was used as the anti-LILRB4 antibody, and clone 6D3C8 (Invitrogen) was used as the anti-LILRB5 antibody. BLI analysis was performed using the BLItz system (ForteBio) according to the attached instructions as follows. Ligands, His-tagged or Fc-fused human LILRB1, LILRB2, LILRB3, LILRB4, or LILRB5, were immobilized on Ni-NTA or AHC sensors (ForteBio), respectively, and unbound proteins were washed with Dulbecco's modified phosphate-buffered saline (DPBS, Gibco). The sensors were loaded with various target proteins or antibodies, allowed to bind, and then immersed in DPBS for dissociation. Curve fitting and data processing using a 1:1 monovalent binding model were performed using BLItz Pro software (ForteBio).

[0071] Since an indirect interaction between mouse LILRB4 and ITG subunit β1 via fibronectin has been reported, we first examined whether human LILRB1, LILRB2, LILRB3, LILRB4, and LILRB5 could directly bind to the ITG β1 subunit by BLI analysis using various LILRB-Fc recombinant proteins. The results are shown in Figure 1A. As shown in Figure 1A, no binding of LILRB3 to the ITG β1 subunit was observed. However, as shown in Figure 1B, LILRB2 and LILRB5 were found to bind to the ITG β1 subunit with high affinity (K = 6.39 ± 2.95 nM) and low affinity (K = 149 ± 106 nM), respectively. LILRB5 bound to the ITG β3 subunit with submicromolar affinity, whereas LILRB1, LILRB3, and LILRB4 did not bind to the ITG β3 subunit. LILRB1 and LILRB4 did not bind to the ITGβ1 subunit. Furthermore, as shown in the top panel of Figure 1A, LILRB2 did not show significant binding to the ITGα5β1 subunit, suggesting that the ITGα5 subunit masks the LILRB2 binding site on the ITGβ1 subunit. These results suggest that LILRB2 and LILRB5 bind to the ITGβ1 subunit with nanomolar or submicromolar affinity, respectively. However, LILRB3 did not bind to the ITGβ1 subunit in this BLI assay, which had an affinity detection limit of approximately 0.5 μM. Furthermore, LILRB5 was found to bind to the ITGβ3 subunit with submicromolar affinity.

[0072] [Example 2] Flow cytometry analysis and cell sorting Whether LILRB2 and LILRB3 co-localize with the ITGβ1 subunit on the cell surface, similar to LILRB4, was examined by the following flow cytometry analysis. First, cryopreserved human peripheral blood mononuclear cells (PBMCs; Cellular Technology) were thawed in a 37°C warm bath, washed, and dead cells were removed using a dead cell removal kit (Miltenyi Biotec). ＋ The cells are CD14 ＋The CD14 was enriched using a MACS sorting column (Miltenyi). ＋ The cell-enriched populations were subjected to flow cytometry or cultured in plastic dishes containing RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Biowest) and 1× antibiotic-antimycotic (ThermoFisher Scientific) at 37°C and 5% CO. ２ The cells were cultured in a humidified atmosphere.

[0073] To prevent nonspecific antibody binding to surface Fcγ receptors, samples were pretreated with 100-fold diluted Human TruStain FcX (BioLegend) on ice for 10 minutes. For surface marker staining, samples were washed with phosphate-buffered saline (PBS) containing 0.5% BSA (blocking buffer) and then stained with 1 × 10 ５ Cells were stained in blocking buffer with unlabeled or fluorochrome-conjugated antibodies, and appropriate fluorescently labeled secondary antibodies, for 15-30 minutes on ice. Samples were analyzed and sorted using a FACSAria III (BD Biosciences). Data were collected using FACS Diva software and analyzed using FlowJo software (Tree Star).

[0074] The flow cytometry analysis showed that CD14 ＋ The expression of LILRB isoforms, ITG subunits, and fibronectin on the surface of monocytes was examined. The results are shown in Figures 2A, 2B, and 2C. As shown in Figure 2A, CD14 ＋ Strong expression of LILRB2, LILRB3, and LILRB4 was observed on the surface of monocytes, whereas expression of LILRB1 or LILRB5 was weak or absent. Expression of ITG subunits was detected at various levels for each of the β1, β2, β3, β7, β8, α4, α5, αV, and αM subunits, as shown in Figure 2B. Fibronectin (FN), which has an N-terminal 30 kDa domain (FN30), interacts with CD14 as shown in Figure 2C. ＋It was suggested that LILRB4 was expressed on monocytes and that a LILRB4-FN-ITG trimeric complex was formed on the cell surface, similar to that observed in mouse peritoneal resident macrophages.

[0075] Example 3 Confocal Laser Scanning Microscope Analysis The distribution of LILRB, ITG subunits, and fibronectin was analyzed using a confocal laser scanning microscope as follows. Cells prepared as in Example 2 were seeded onto glass-bottom dishes (polylysine-coated or uncoated, dish diameter 35 mm, glass diameter 14 mmφ, glass thickness No. 1S / 1.5 (0.16-0.19 mm); #D11131H; Matsunami Co., Ltd.) and cultured for 1 hour. The resulting cells were fixed with 0.5-2% paraformaldehyde (PFA) at room temperature for 1 hour. After washing the cells three times with a washing solution (PBS containing 1% BSA), the fixed cells were double-stained with a monoclonal antibody against LILRB2, LILRB3, or LILRB4 and an antibody against the ITG β1 subunit. After thorough washing, the cells were mounted in a small amount of SlowFade (Invitrogen) and analyzed in two dimensions using a Leica SP8 confocal microscope system.

[0076] Next, the correlation coefficient r between the two sets of observation parameters, i.e., the ITG and LILRB fluorescence signals, was evaluated by Pearson correlation analysis, as described in Adler et al. (Cytometry A. 2010, 77:733) and Ito et al. (Int Immunol. 2022 Jul 26;34(8):435-444). Each signal profile of the cell outline was analyzed using Leica SP8 software, and the r value was calculated. The r values ​​were obtained for more than 20 randomly selected cells from the captured images and displayed graphically. The average r value was interpreted according to the following criteria:

[0077] r = -1: Perfect negative linear relationship -1 < r ≦ -0.70: Strong negative linear relationship = 0.7 < r ≦ = 0.4: Negative relationship -0.4 < r ≦ -0.2: Weak negative linear relationship -0.2 < r ≦ +0.20: No significant relationship +0.2 < r ≦ +0.4: Weak positive relationship +0.4 < r ≦ +0.7: Positive linear relationship +0.7 < r < +1: Strong positive linear relationship r = +1: Perfect positive linear relationship r > +0.2: Positive correlation

[0078] The captured images are shown in Figure 3A, and the Pearson's correlation coefficients r are shown in Figure 3B. As shown in Figure 3B, the Pearson's correlation coefficients r calculated for the fluorescent signals at the focal plane of cell adhesion were positive (r > 0.2) for all combinations, regardless of whether the dish was FN-uncoated or FN-coated, suggesting spatial proximity between LILRB2, LILRB3, and LILRB4 and the ITGβ1 subunit.

[0079] [Example 4] Interaction between anti-LILRB3 antibody and anti-ITGβ1 subunit antibody in binding to target As shown in Figure 3A of Example 3, during the confocal scanning microscope analysis, double staining of LILRB3 and ITGβ1 subunit frequently resulted in a decrease in the fluorescent signal of the ITGβ1 subunit (Figure 3A, second row). To verify this, competition between anti-LILRB3 antibody and anti-ITGβ1 subunit antibody was investigated as follows. Monocytes or RAW264.7 cells (5 x 10 ４Cells (100 cells / tube) were stained simultaneously with either directly fluorescently labeled or unlabeled anti-ITGβ1 subunit antibody and anti-LILRB3 antibody at 0.5 μg / tube each on ice for 1 hour, or by sequential addition of either antibody for 30 minutes followed by 30 minutes of staining. The cells were then washed, and if necessary, a fluorescently labeled second antibody was added and stained for another 30 minutes. After washing, the cells were subjected to flow cytometry analysis. For the staining process, a group containing an appropriate isotype antibody corresponding to the anti-ITGβ subunit antibody or anti-LILRB3 antibody was used as a comparison. Histograms of each cell population were generated using FlowJo software, and the mean fluorescence intensity was calculated. The level of fluorescence intensity obtained with the anti-ITGβ1 subunit antibody, relative to 100%, was plotted as a percentage control, with the level of fluorescence obtained with the anti-LILRB antibody representing the control. The results are shown in Figures 4A to 4C. When monocytes were stained individually with either the anti-LILRB3 monoclonal antibody or the anti-ITGβ1 subunit antibody, clear, bright fluorescent signals were detected for both antibodies, as shown in the top row of Figure 4A. Simultaneous staining of monocytes with these antibodies reduced the fluorescent signal of the ITGβ1 subunit, as shown in the second row of Figure 4A. Staining first with the anti-ITGβ1 subunit antibody and then with the anti-LILRB3 monoclonal antibody yielded a bright signal of the ITGβ1 subunit, as shown in the bottom row of Figure 4A. These confocal microscopy observations were also reproduced by flow cytometry, as shown in Figure 4B. Further flow cytometry measurements and statistical analysis were performed using two different commercially available LILRB3 monoclonal antibodies, #222821 and #08, and monocyte preparations from three different specimens of cryopreserved human PBMCs. The results are shown in Figure 4C.

[0080] As shown in Figure 4C, the ability of the anti-LILRB3 monoclonal antibody to interfere with anti-ITG β1 subunit antibody binding varied between the two antibodies and possibly among the three different monocyte samples, but both #222821 and #08 antibodies reproducibly exhibited interference effects. These results suggest that the close spatial relationship or neighboring relationship between LILRB3 and the ITG β1 subunit results in the inhibition of target binding by the anti-LILRB3 monoclonal antibody.

[0081] Example 5: Interference of monoclonal antibodies against the ITG β subunit-LILRB combination As shown in Example 4, LILRB3 and the ITG β1 subunit are spatially close to each other. Therefore, using flow cytometry, we investigated the possibility of interference with anti-LILRB3 monoclonal antibody #08 for other anti-ITG β subunit antibodies and anti-ITG α subunit antibodies. The results are shown in Figures 5A to 5D. As shown in Figures 5A and 5B, anti-LILRB3 monoclonal antibody #08 did not exhibit inhibitory activity against the ITG subunit monoclonal antibodies for the β2 subunit, α4 subunit, α5 subunit, and αM subunit.

[0082] Furthermore, the proximity relationship between LILRB2 and the ITGβ subunit was investigated by antibody interference in the same manner as described above, and the results showed that the only combination investigated was competition between the anti-LILRB2 monoclonal antibody and the anti-ITGβ1 subunit antibody, as shown in Figures 5C and 5D. The above confocal scanning microscopy analysis and monoclonal antibody interference analysis confirmed that LILRB3 and the ITGβ1 subunit are in a close spatial relationship or neighboring relationship on the surface of monocytes, and that there is a neighboring relationship or direct binding between LILRB2 and the ITGβ1 subunit as observed in the BLI analysis.

[0083] Example 6 Measurement of the Spatial Relationship between LILRB and the ITGβ Subunit by Fluorescence Resonance Energy Transfer (FRET) Using a Confocal Laser Scanning Fluorescence Microscope To more precisely evaluate the close spatial relationship or neighboring relationship between LILRB and the ITGβ subunit, as indicated by antibody-antibody interference, fluorescence resonance energy transfer (FRET) was measured using a confocal laser scanning fluorescence microscope. The FRET efficiency (%) was calculated as follows: Fluorescence generated by laser excitation light of a wavelength given to the donor fluorescent dye is absorbed as excitation energy by the fluorescent dye on the acceptor side, which is located at a distance of 2 to 10 nm, resulting in fluorescence quenching. When the fluorescence of the acceptor fluorescent dye is quenched (bleached) by irradiating it with strong excitation light, the fluorescence of the donor side is not absorbed by the acceptor, resulting in an increase in fluorescence intensity. By measuring the fluorescence intensity of the donor before and after bleaching, the FRET efficiency can be calculated using the following formula: FRET efficiency (%) = (Dpost - Dpre) / Dpost × 100, where Dpre and Dpost are the fluorescence intensities before and after bleaching, respectively. In the above calculation results, a FRET efficiency higher than 5% was considered significant. Figure 6A shows fluorescence microscopy images before and after bleaching when the donor was an LTGβ1 subunit antibody and the acceptor was an anti-LILRB3 antibody. Figure 6B shows the FRET efficiency on FN-coated and uncoated plates. As shown in Figures 6A and 6B, when the fluorescent dye of the anti-ITGβ1 antibody was used as the donor and the fluorescent dye of the anti-LILRB3 antibody as the acceptor, significant FRET was observed at the plate adhesion surface of monocytes seeded on FN-coated and uncoated plates, i.e., at focal adhesions. Furthermore, as shown in Figures 6C and 6D, similar significant FRET was observed between LILRB2 and the ITGβ1 subunit.From the above results, it was revealed that the distance between LILRB3 or LILRB2 and the ITGβ1 subunit is 2 to 10 nm, at which FRET is believed to occur, which is comparable to the size of a typical protein molecule, and therefore LILRB3 and the ITGβ1 subunit, and LILRB2 and the ITGβ1 subunit, are adjacent to each other or directly bound to each other.

[0084] Example 7 Enhancement of Syk kinase phosphorylation by treatment of monocytes with anti-LILRB2 or anti-LILRB3 antibodies Following cell adhesion, activation of integrins at focal adhesions induces activation of Src family kinases immediately downstream, initiating tyrosine phosphorylation of FAK and Syk, leading to canonical and pro-inflammatory signaling, respectively. It has been reported that tyrosine phosphorylation of Syk kinase is enhanced in LILRB4-deficient cells adhered to FN-coated plates in mouse macrophages and human THP-1 monocytic leukemia cells. In this study, we demonstrated that this signaling is mediated by human CD14 ＋ Whether this also occurs with LILRB2, LILRB3, and LILRB4 on monocytes was examined as follows.

[0085] Monocytes were treated with the appropriate antibody, plated on FN-coated or non-coated dishes, and incubated for 1 hour to prepare cell lysates for Western blot analysis. The cell lysates were then used for the Western blot analysis described below. For FN immobilization, human FN solution was diluted to 10 μg / ml with PBS and added to a 24- or 48-well culture plate. The plate was incubated at room temperature for 2 hours, and the plate was washed twice with PBS before use. Cells were mixed with the antibody and plated in FN-coated or non-coated wells at a concentration of 2.0 × 10 cells. ５Cells were seeded at 1000 x g / well and incubated at 37°C for 1 hour. Cells were lysed in RIPA lysis buffer (1% NP-40, 25 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and proteinase inhibitors) containing PhosphoStop (Sigma-Aldrich). The protein content of the lysate was measured by the BCA method, and the lysate was subjected to SDS-PAGE electrophoresis under reducing conditions on a 10% polyacrylamide gel (Wako Pure Chemical Industries, Ltd.), transferred to a PVDF membrane, and then immunoblotted with CanGet signal solution. Fluorescence detection was performed using Amersham ECL Prime (Cytiva) and ImageQuant LAS-4000 (Cytiva), and the results are shown in Figures 7A and 7B.

[0086] As shown in Figures 7A and 7B, enhancement of Syk phosphorylation was observed not only with anti-LILRB3 monoclonal antibody but also with anti-LILRB2 monoclonal antibody, and the effect was comparable to that of anti-LILRB4 monoclonal antibody ZM4.1. Furthermore, as shown in Figure 7B, the combinations of anti-LILRB2 monoclonal antibody and anti-LILRB3 monoclonal antibody, anti-LILRB2 monoclonal antibody and anti-LILRB4 monoclonal antibody, and anti-LILRB3 monoclonal antibody and anti-LILRB4 monoclonal antibody did not significantly enhance the increase in Syk phosphorylation. On the other hand, the FAK phosphorylation response to these antibodies was not as pronounced as that of Syk phosphorylation. Based on the above, it is thought that anti-LILRB2 antibodies and anti-LILRB3 antibodies at least functionally dissociate the direct binding between LILRB2 and the ITGβ1 subunit and the adjacent or direct binding between LILRB3 and the ITGβ1 subunit, respectively, leading to enhanced ITG-induced pro-inflammatory Syk activation.

[0087] Example 8: Changes in cell morphology and cytokine release in monocytes treated with anti-LILRB antibodies The effects of cell treatment with anti-LILRB2 and anti-LILRB3 antibodies on cellular responses, such as changes in cell morphology and cytokine release, were examined. TNF-α in the cell culture supernatant was measured using an ELISA Max Standard TNF-α Set (BioLegend) according to the attached protocol. Absorbance was read at 450 nm using a Model 680 microplate reader (Bio-Rad). IL-6 in the cell culture supernatant was measured by ELISA using recombinant human IL-6 protein as a standard and an anti-human IL-6 antibody (both from eBioscience) in the same manner as for the TNF-α measurement described above. Absorbance was read at 450 nm using a Model 680 microplate reader (Bio-Rad). The results are shown in Figures 8A and 8B. As shown in Figure 8A, cells cultured overnight in the presence of any of the anti-LILRB2 monoclonal antibody, anti-LILRB3 monoclonal antibody, and anti-LILRB4 monoclonal antibody showed elongation compared to cells treated with an isotype antibody. Furthermore, as shown in Figure 8B, it was found that the TNF-α and IL-6 levels in the culture supernatant were increased. These results indicate that treatment of cells with anti-LILRB2 monoclonal antibody and anti-LILRB3 monoclonal antibody increases CD14 ＋ It was suggested that monocytes were activated.

[0088] Example 9: Verification using mouse PIR-B, a functional homolog of human LILRB3. Because the LILRB family lineup differs between mice and humans, it is difficult to examine the in vivo effects of blocking antibodies against LILRB2 and LILRB3. Given that the physiological ligands of LILRB4 and LILRB1 / LILRB2 in mice are FN and MHC class I, respectively, gp49B and PIR-B are thought to be orthologs of human LILRB4 and LILRB1 / LILRB2, respectively. Because the amino acid sequences of the mature ITGβ1 subunit proteins of humans and mice are 93.8% identical, showing high similarity, we investigated whether mouse PIR-B can bind to the human ITGβ1 subunit in the same way as human LILRB2. As shown in Figure 9A, BLI analysis did not reveal direct binding of PIR-B to the human ITGβ1 subunit. However, as shown in Figure 9B, PIR-B and the ITGβ2 subunit expressed in the mouse macrophage cell line RAW264.7 were stained with fluorescently labeled antibodies and images were captured using a confocal laser scanning fluorescence microscope. The Pearson's correlation coefficient (r) measured for the fluorescent signals at the focal plane of cell adhesion was positive (r > 0.2) for the combination of PIR-B and the ITGβ2 subunit, regardless of whether the dish was FN-coated or uncoated, suggesting spatial proximity between PIR-B and the ITGβ2 subunit. Therefore, the degree of coexistence of PIR-B and the ITGβ2 subunit on cells shared a property similar to that of the relationship between LILRB3 and ITGβ1.

[0089] On the other hand, it was reported in 2004 that PIR-B can inhibit ITG signaling in mouse monocytes and neutrophils (J Immunol. 2004 Nov 1;173(9):5757-65.), but the mechanism by which PIR-B inhibits ITG signaling was unknown. This example unexpectedly demonstrated that human LILRB3, which is homologous to PIR-B at the genetic level, is spatially adjacent to or directly binds to ITG, and further revealed that PIR-B and mouse ITG colocalize. This suggests that PIR-B may also be a functionally homologous molecule to human LILRB3. To verify this, we performed a monoclonal antibody interference assay using PIR-B and ITG β1 and β1 subunits expressed in the mouse macrophage cell line RAW264.7. The results are shown in Figures 10A and 10B.

[0090] As shown in Figures 10A and 10B, the anti-PIR-A / B domain D1D2 (the first and second N-terminal domains of the six immunoglobulin-like domains in PIR) antibody 6C1 and the anti-PIR-A / B domain D5D6 (the fifth and sixth N-terminal domains of the six immunoglobulin-like domains in PIR) antibody 10.1 did not show significant competition with the anti-ITGβ1 antibody (rabbit polyclonal antibody; manufactured by GTX). However, another anti-PIR-A / B domain D5D6 antibody, 11.3, significantly competed with the anti-ITGβ1 antibody. Furthermore, as shown in Figure 10C, neither the anti-PIR-A / B domain D1D2 antibody 6C1 nor the anti-PIR-A / B domain D5D6 antibody 11.3 competed with the anti-ITGβ2 subunit monoclonal antibody (clone M18 / 2). These results suggest that the previously reported ITG signaling regulation by PIR-B is actually due to the spatial proximity of PIR-B and the ITGβ subunit, resulting from direct or indirect binding in mouse monocytes and neutrophils. Therefore, mouse PIR-B may be a functional homolog of human LILRB3. Furthermore, as shown in Figures 11A and 11B, stimulation of mouse peritoneal cells with the PIR-A / B domain D5D6 antibody 11.3 resulted in enhanced Syk phosphorylation, demonstrating that this antibody can functionally dissociate the binding between PIR-B and the ITGβ subunit. These findings suggest that the in vivo function of human LILRB3 can be assessed by examining the ITG signaling regulation function of PIR-B in in vivo mouse models, such as those involving tumor loading. For example, it has been reported that when PIR-B is not functional in myeloid-derived suppressor cells (MDSCs) that infiltrate cancer tissue, cancer immunity is enhanced (Immunity. 2011 Mar 25;34(3):385-95.), therefore, antibodies that inhibit the interaction between LILRB3 and ITG are thought to be effective in cancer treatment.

Claims

1. An immune checkpoint inhibitor containing as an active ingredient a substance that inhibits the interaction between integrin and immunoinhibitory receptor (hereinafter referred to as LILR) B3.

2. The immune checkpoint inhibitor of claim 1, wherein the interaction between the integrin and LILRB3 includes interaction with the integrin β1 subunit.

3. The immune checkpoint inhibitor according to claim 1 or 2, wherein the substance that inhibits the interaction between integrin and LILRB3 is an anti-LILRB3 antibody or a derivative thereof.

4. A therapeutic agent for immune checkpoint-related diseases, comprising as an active ingredient a substance that inhibits the interaction between integrin and LILRB3.

5. The therapeutic agent for an immune checkpoint-associated disease according to claim 4, wherein the immune checkpoint-associated disease is selected from the group consisting of autoimmune diseases, cancer, inflammatory diseases, Alzheimer's disease, infectious diseases, and allergic diseases.

6. The therapeutic agent for an immune checkpoint-associated disease according to claim 4 or 5, wherein the interaction between the integrin and LILRB3 includes the interaction between the integrin β1 subunit and LILRB3.

7. The therapeutic agent for immune checkpoint-related diseases according to claim 4 or 5, wherein the substance that inhibits the interaction between integrin and LILRB3 is an anti-LILRB3 antibody or a derivative thereof.

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