Anti-human immunosuppressive receptor lilrb4 monoclonal antibodies or fragments thereof, immune checkpoint inhibitors, therapeutic agents for immune checkpoint-related diseases, and methods for measuring human immunosuppressive receptor lilrb4

Abstract

The present invention provides anti-human LILRB4 monoclonal antibodies or fragments thereof, immune checkpoint inhibitors, therapeutic agents for immune checkpoint-related diseases, and methods for measuring human LILRB4.SOLUTION: Anti-human immunosuppressive receptor LILRB4 monoclonal antibodies or fragments thereof that specifically bind to a human immunosuppressive receptor LILRB4 and inhibit binding between fibronectin and the human immunosuppressive receptor LILRB4, immune checkpoint inhibitors containing the anti-human immunosuppressive receptor LILRB4 monoclonal antibodies or fragments thereof as active ingredients, therapeutic agents for immune checkpoint-related diseases, and methods for measuring a human immunosuppressive receptor LILRB4 in a biological sample using the anti-human immunosuppressive receptor LILRB4 monoclonal antibodies or fragments thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or an antibody fragment thereof, an immune checkpoint inhibitor, a therapeutic agent for immune checkpoint-related diseases, and a method for measuring the human immunoinhibitory receptor LILRB4. [Background technology]

[0002] In recent years, cancer immunotherapy using immune checkpoint inhibitors has attracted attention. Immune checkpoint inhibitors are drugs that bind to immune checkpoint molecules or their ligands, which have the effect of suppressing immune responses against the self and suppressing excessive immune responses, and inhibit the transmission of immunosuppressive signals, thereby relieving the suppression of activation of T cells and other immune system cells by immune checkpoint molecules.

[0003] The present inventors have discovered that inflammatory diseases caused by infection or autoimmunity can be diagnosed by using LILRB4 (Leukocyte Ig-like receptor B4, hereinafter also referred to as B4), an immunosuppressive receptor for an unknown ligand (Patent Document 1).

[0004] Although CD166, ApoE, Angptls, etc. have been reported as ligands of LILRB4 (Non-Patent Documents 1 to 3), it has not been sufficiently proven that these are physiological ligands. The present inventors have discovered that the physiological ligand of B4 is fibronectin, and have reported that a substance that inhibits the binding of B4 to fibronectin is effective in treating immune checkpoint-related diseases (Patent Document 2).

[0005] Fibronectin (FN) is a glycoprotein of approximately 259 kDa present in the extracellular matrix (ECM), on cell surfaces, and in body fluids. Fibronectin can be separated into six domains by treatment with the proteolytic enzyme thermolysin. These domains are designated based on their specific molecular binding abilities: 1. fibrin / heparin binding domain (FN), 2. collagen binding domain (FN), 3. heparin binding domain (FN), 4. cell / integrin-binding domain (CBD) (FN), 5. second heparin binding domain (FN), and 6. second fibrin binding domain (FN). Thus, FN is composed of multiple domains with different binding abilities to physiological molecules. In some autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), changes in plasma and body fluids (e.g., synovial fluid) FN concentrations have been observed. Furthermore, domain analysis, which evaluates FN fragmentation using monoclonal antibodies, has been reported to be useful for diagnosing and assessing disease severity (Non-Patent Documents 4-6). In particular, fibrin / heparin-binding FN concentrations were 24±12 μg / ml (p<0.003) and 36±22 μg / ml (p<0.00002) in SLE and RA, respectively, compared with healthy controls (61±18 μg / ml), suggesting potential diagnostic applications. Furthermore, FN has been reported to promote the metastatic and invasive potential of lung cancer cell lines (Non-Patent Document 7).

[0006] Although anti-mouse gp49A / B antibody is known as a monoclonal antibody against mouse B4, no monoclonal antibody against human B4 has been reported that has the activity to inhibit the binding of FN to B4. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2018-25554 [Patent Document 2] International Publication No. 2021 / 029318 [Non-patent literature]

[0008] [Non-Patent Document 1] J Immunol. 2018 Feb 1;200(3):1207-1219. [Non-patent document 2] Blood. 2014 Aug 7;124(6):924-935. [Non-patent document 3] Nature. 2018 Oct;562(7728):605-609. [Non-patent document 4] Rheumatology (Oxford). 2007 Jul;46(7):1071-1075. [Non-patent document 5] J Rheumatol. 1987 Oct;14(5):1052-1054. [Non-patent document 6] Rheumatol Int. 2013 Jan;33(1):37-43. [Non-Patent Document 7] Br J Cancer 2009 Jul 21;101(2):327-334. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide an anti-human LILRB4 monoclonal antibody or antibody fragment thereof that specifically binds to human LILRB4 and inhibits the binding of human LILRB4 to fibronectin, an immune checkpoint inhibitor, a therapeutic agent for immune checkpoint-related diseases, and a method for measuring human LILRB4. [Means for solving the problem]

[0010] The present inventors have produced an anti-human immunoinhibitory receptor LILRB4 monoclonal antibody that specifically binds to human LILRB4 and inhibits the binding of human LILRB4 to its physiological ligand, FN, and have found that this monoclonal antibody is useful as a therapeutic agent for immune checkpoint-related diseases, thereby completing the present invention. The present invention includes the following aspects. [1] An anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or an antibody fragment thereof that specifically binds to the human immunoinhibitory receptor LILRB4 and inhibits the binding of fibronectin to the human immunoinhibitory receptor LILRB4. [2] The anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or antibody fragment thereof described in [1], wherein the fibronectin binds to the immunoinhibitory receptor LILRB4 via the amino acid sequence represented by sequence number 2 in the fibronectin. [3] An immune checkpoint inhibitor containing the anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or its antibody fragment described in [1] or [2] as an active ingredient. [4] A therapeutic agent for immune checkpoint-related diseases, comprising the anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or its antibody fragment according to [1] or [2] as an active ingredient. [5] The therapeutic agent according to [4], wherein the immune checkpoint-related disease is selected from the group consisting of autoimmune diseases, cancer, inflammatory diseases, and allergic diseases. [6] The therapeutic agent according to [5], wherein the cancer is a primary cancer. [7] A method for measuring the human immunoinhibitory receptor LILRB4 contained in a biological sample, using the anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or its antibody fragment described in [1] or [2]. [Effects of the Invention]

[0011] According to the present invention, there can be provided an anti-human immunoinhibitory receptor LILRB4 monoclonal antibody (hereinafter also referred to as anti-human LILRB4 monoclonal antibody or anti-human B4 monoclonal antibody) or an antibody fragment thereof that specifically binds to human LILRB4 and inhibits the binding of human LILRB4 to fibronectin, an immune checkpoint inhibitor, a therapeutic agent for immune checkpoint-related diseases, and a method for measuring human LILRB4. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows the results of ELISA testing the binding activity of the anti-human LILRB4 monoclonal antibody-containing culture supernatant obtained in Example 2 to various leukocyte Ig-like receptors (LILRs). In the figure, gp49B represents recombinant mouse LILRB4. [Figure 2] Figure 1 shows the results of flow cytometry analysis of the binding of anti-human LILRB4 antibodies to ligands (fibronectin and ApoE) in Example 3. In the figure, "Unstimulated" indicates the results of flow cytometry analysis of culture supernatants containing anti-human LILRB4 antibodies (#25, #10, #11) in the absence of FN30-Fc and ApoE, "FN30Fc(PB)" indicates the presence of FN30-Fc, "ApoE(SP)" indicates the presence of ApoE, and "FN30Fc(PB)&ApoE(SP)" indicates the results of flow cytometry analysis of culture supernatants containing anti-human LILRB4 antibodies (#25, #10, #11) in the presence of FN30-Fc and ApoE. In the figure, "No Ab" indicates the results when medium containing no antibody was added. [Figure 3]This shows the GFP fluorescence intensity upon addition of culture supernatant containing the anti-human LILRB4 antibodies (#1, #5, #8, #10, #11, #25, and #31) of Example 3. In the figure, "Unstimulated" indicates the GFP fluorescence intensity in the absence of FN30-Fc and ApoE, "FN30Fc(PB)" indicates the GFP fluorescence intensity in the presence of FN30-Fc, "ApoE(SP)" indicates the GFP fluorescence intensity in the presence of ApoE, and "FN30Fc(PB)&ApoE(SP)" indicates the GFP fluorescence intensity in the presence of FN30-Fc and ApoE. [Figure 4] The graph shows the geometric mean of GFP fluorescence intensity upon addition of culture supernatant containing anti-human LILRB4 antibodies (#1, #5, #8, #10, #11, #25, and #31) from Example 3. In the graph, "Unstimulated" indicates GFP fluorescence intensity in the absence of FN30-Fc and ApoE, "FN30Fc(PB)" indicates GFP fluorescence intensity in the presence of FN30-Fc, "ApoE(SP)" indicates GFP fluorescence intensity in the presence of ApoE, and "FN30Fc(PB)&ApoE(SP)" indicates GFP fluorescence intensity in the presence of FN30-Fc and ApoE. DETAILED DESCRIPTION OF THE INVENTION

[0013] [Anti-human LILRB4 monoclonal antibody and its antibody fragment] The anti-human LILRB4 monoclonal antibody of the present invention specifically binds to human LILRB4 and inhibits the binding of human LILRB4 to fibronectin. Human LILRB4 including a signal sequence consists of the amino acid sequence shown in SEQ ID NO: 1. The human LILRB4 signal sequence stretches from methionine at position 1 to alanine at position 23 in SEQ ID NO: 1, and mature human LILRB4 consists of the amino acid sequence from glycine at position 24 to tryptophan at position 258 in the amino acid sequence shown in SEQ ID NO: 1. Fibronectin can bind to human LILRB4 via the amino acid sequence shown in SEQ ID NO: 2 in fibronectin. In other words, the amino acid sequence shown in SEQ ID NO: 2 is the target sequence in fibronectin for human LILRB4.

[0014] Monoclonal antibodies can be produced according to known monoclonal antibody production methods, such as those described in "Monoclonal Antibodies," co-authored by Nagamune Kaoru and Terada Hiroshi, 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 Mar. 12; 356 152-154 and J. Immunol. Methods Mar. 1; 249 147-154.

[0015] The antigen used to generate anti-human LILRB4 monoclonal antibodies can be the human LILRB4 protein, a partial fragment (peptide) thereof, or a vector incorporating cDNA encoding the human LILRB4 protein. To obtain monoclonal antibodies that recognize the higher-order structure of human LILRB4, the optimal immunization antigen gene is a full-length human LILRB4 vector, a construct containing the full-length human LILRB4 gene. Constructs incorporating a partial region of the human LILRB4 sequence can also be used as immunization antigen genes. A preferred partial region of the human LILRB4 sequence is the region of human LILRB4 that binds to human LILRB4 (fibronectin-binding site), which is the amino acid sequence represented by SEQ ID NO: 2, the target sequence of human LILRB4 in fibronectin. DNA immunization can be performed by subcutaneously injecting the above gene constructs, either alone or in combination, into animals (e.g., mice or rats) using various gene transfer methods (e.g., intramuscular injection, electroporation, gene gun, etc.) and allowing them to be incorporated into the cells.

[0016] Examples of the antibody fragment include F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, mutants thereof, fusion proteins or fusion peptides containing an antibody portion, etc. The antibody fragment can be produced according to known methods for producing antibody fragments.

[0017] As used herein, the term "specifically binds" by an antibody means that the antibody binds to human LILRB4 without substantially binding to other different polypeptides.

[0018] The anti-human LILRB4 monoclonal antibody can be further purified before use, if necessary. Methods for purifying and isolating the anti-human LILRB4 monoclonal antibody include conventionally known methods, such as salting out (e.g., ammonium sulfate precipitation), gel filtration using Sephadex or the like, ion exchange chromatography, and affinity purification using a protein A column or the like.

[0019] The anti-human LILRB4 monoclonal antibody or antibody fragment thereof of the present invention can inhibit the binding of fibronectin to human LILRB4 via the amino acid sequence in fibronectin shown in SEQ ID NO:2.

[0020] Inhibition of the binding of fibronectin to human LILRB4 can be assessed by assessing inhibition of fibronectin binding to cells expressing human LILRB4, including, but not limited to, spleen cells, peripheral blood leukocytes, bone marrow cells, and B cells isolated therefrom, plasma cells, monocytes / macrophages, dendritic cells, eosinophils, basophils, neutrophils, mast cells, and activated T cells.

[0021] The nucleotide sequence and amino acid sequence of LILRB4 can be found in the database provided by the National Center for Biotechnology Information (NCBI). Examples of human (Homo sapiens) LILRB4 include Entrez GeneID 11006 (as of June 17, 2019) and RefSeq ProteinIDs NP_001265355.2, NP_001265356.2, NP_001265357.2, NP_001265358.2, and NP_001265359.2 (corresponding to isoforms 1 to 5).

[0022] The nucleotide sequence and amino acid sequence of fibronectin can be found in the database provided by the National Center for Biotechnology Information (NCBI). Examples of human (Homo sapiens) fibronectin include Entrez GeneID 2335 and RefSeq ProteinIDs NP_997647, NP_001352447, and XP_005246463.

[0023] Fibronectin activates immune checkpoint molecules by binding to LILRB4 present on the cell surface of plasma cells, T cells, macrophages, etc. via the amino acid sequence in fibronectin shown in SEQ ID NO: 2. In other words, LILRB4 exerts an immunosuppressive function by binding to fibronectin via the amino acid sequence in fibronectin shown in SEQ ID NO: 2.

[0024] [Immune checkpoint inhibitors] The immune checkpoint inhibitor of the present invention comprises the anti-human LILRB4 monoclonal antibody of the present invention or an antibody fragment thereof as an active ingredient, and may further comprise pharmaceutically acceptable carriers and additives.

[0025] 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.

[0026] The immune checkpoint inhibitor 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).

[0027] The immune checkpoint inhibitor of the present invention can be used as a therapeutic agent for immune checkpoint-related diseases. The dose of the immune checkpoint inhibitor 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.

[0028] [Therapeutic agent for immune checkpoint-related diseases] The therapeutic agent for immune checkpoint-related diseases of the present invention contains the human LILRB4 monoclonal antibody of the present invention or an antibody fragment thereof as an active ingredient. In the present invention, immune checkpoint-associated diseases are not particularly limited as long as they involve the immune checkpoint molecule LILRB4, and examples include autoimmune diseases, cancers, inflammatory diseases, and allergic diseases.

[0029] 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, and glomerulonephritis. Examples of cancer include lung cancer, colon cancer, stomach cancer, kidney cancer, malignant melanoma, Hodgkin's lymphoma, head and neck cancer, pancreatic cancer, liver cancer, prostate cancer, osteosarcoma, leukemia, etc. Cancer may be primary or metastatic, but the method is preferably used for primary cancer.

[0030] 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, Sjögren's syndrome, Behçet'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.

[0031] Allergic diseases include allergic rhinitis, bronchial asthma, urticaria / atopic dermatitis, shingles, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergies, 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, organ transplant rejection, and graft-versus-host disease (GVHD).

[0032] 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.

[0033] The therapeutic agent for immune checkpoint-associated diseases of the present invention can be in various forms, such as liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, etc. A preferred embodiment is an injection, which is preferably administered parenterally (e.g., intravenously, transdermally, intraperitoneally, intramuscularly).

[0034] 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.

[0035] [Method for measuring human LILRB4] The method for measuring human LILRB4 of the present invention is a method for measuring human LILRB4 contained in a biological sample using the anti-LILRB4 monoclonal antibody of the present invention or an antibody fragment thereof. Examples of the method for measuring human LILRB4 of the present invention include an immunological method for measuring human LILRB4 in a biological sample, which comprises reacting human LILRB4 in the biological sample with the anti-human LILRB4 monoclonal antibody or antibody fragment thereof of the present invention, adding a labeled antibody or labeled antibody fragment in which a label is bound to the anti-human LILRB4 monoclonal antibody or antibody fragment thereof, forming an immune complex consisting of human LILRB4, the anti-human LILRB4 monoclonal antibody or antibody fragment thereof, and the labeled antibody or labeled antibody fragment, and measuring the amount of label in the formed immune complex.

[0036] In the measurement method of the present invention using the anti-human LILRB4 monoclonal antibody or antibody fragment thereof, examples of biological samples include blood, such as serum, plasma, or whole blood, lymph, interstitial fluid, cerebrospinal fluid, body cavity fluid, digestive fluid, nasal mucus, tears, sweat, and urine, from animals including humans. Examples of biological samples also include tissue fragments derived from organs, such as the lungs, stomach, and kidneys of humans, or thin slices thereof. The biological sample may be the biological sample collected directly from a subject, or may be a sample that has been subjected to a commonly performed treatment, such as dilution or concentration, of a collected biological sample. The biological sample may be collected or prepared at the time of carrying out the measurement method, or may be collected or prepared in advance and stored.

[0037] The immunological measurement methods are classified into enzyme immunoassays (EIA or ELISA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), fluorescence polarization immunoassays (FPIA), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassays, immunohistochemical staining, etc., depending on the label of the labeled detection antibody. Any of these can be used in the measurement method of the present invention, but ELISA is preferred for liquid phase samples, and immunohistochemical staining is preferred for solid samples such as tissue sections, as they allow for simple and rapid measurement of the target substance.

[0038] After washing the immune complex, human LILRB4 in the biological sample can be measured by measuring the label in the immune complex. For example, in the case of ELISA, human LILRB4 in the biological sample can be measured by reacting the labeled enzyme with the enzyme's substrate and measuring the absorbance of the colored product (sandwich method). Alternatively, human LILRB4 in the biological sample can be measured by reacting the anti-human LILRB4 monoclonal antibody or antibody fragment thereof of the present invention immobilized on a solid support with human LILRB4 in the biological sample, adding an unlabeled anti-human LILRB4 monoclonal antibody or antibody fragment thereof (primary antibody), further adding an enzyme-labeled labeled secondary antibody (secondary antibody) against the unlabeled antibody or antibody fragment thereof, and measuring the label of the secondary antibody. Alternatively, human LILRB4 in the biological sample can be measured by labeling the secondary antibody with biotin, binding avidin or streptavidin labeled with an enzyme or the like to the biotin, labeling the secondary antibody with an enzyme or the like, and measuring the label of the secondary antibody.

[0039] Alternatively, human LILRB4 in a biological sample can be measured by adding an unlabeled anti-human LILRB4 monoclonal antibody or its antibody fragment (primary antibody) to human LILRB4 immobilized on a solid support or a conjugate of human LILRB4 and a protein such as BSA to form an immune complex consisting of human LILRB4 and the primary antibody on the solid support, followed by adding a biological sample and further adding a labeled secondary antibody (secondary antibody) against this unlabeled antibody, and measuring the label of the labeled secondary antibody (competitive method).

[0040] The solid support is not particularly limited as long as it can stably support an antibody or antibody fragment. Preferred materials for the solid support include polymeric materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, nitrocellulose, cellulose acetate, cellulose acetate, and polyethylene terephthalate, as well as glass, ceramics, magnetic particles, and metals. Preferred shapes of the solid support include tubes, beads, plates, fine particles such as latex, and sticks.

[0041] The label may be an enzyme such as peroxidase or alkaline phosphatase in ELISA, or may be a label such as 125 I, 131 I, 35 S, 3 Radioactive substances such as H can be used for the FPIA method; fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials can be used for the CLIA method; enzymes such as luciferase and β-galactosidase, luminescent substrates that are converted into luminescent substances by the respective enzymes, and luminescent substances such as luciferin and aequorin can also be used as labels. In addition, nanoparticles such as gold colloids and quantum dots can also be used as labels.

[0042] In ELISA, the substrate of the labeled enzyme can be 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), O-phenylenediamine (OPD), etc. if the enzyme is peroxidase, and p-nitrophenyl phosphate (pNPP), etc. if the enzyme is alkaline phosphatase. [Example]

[0043] 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.

[0044] [Example 1] Production of recombinant human LILRB4 The cDNA of the extracellular domain of human LILRB4 (from glycine at position 24 to glycine at position 219; hereinafter also referred to as exB4) in which the isoleucine at position 156 of human LILRB4, which has the amino acid sequence shown in SEQ ID NO: 1 and in which the histidine at position 166 of human LILRB4 has been replaced with cysteine, was chemically synthesized and inserted into the pUCIDT-KAN vector (Integrated DNA Technologies) to construct the plasmid pUCIDT-KAN / exB4(I156C / H166C). This plasmid was digested with NdeI and XhoI, and the resulting B4 cDNA fragment was ligated into the NdeI / XhoI sites of pET-26M, a modified version of pET-26b(+) (Novagen) in which the NdeI-NcoI site had been replaced with Gly-x6 His tag-Ala. The resulting plasmid, pET-26M / exB4(I156C / H166C), was transfected into E. coli BL21(DE3), and expression of the B4 protein was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to the medium. After culturing, the cells were suspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 8.0), sonicated, and centrifuged to collect inclusion bodies containing exB4. The resulting inclusion bodies were solubilized in denaturation buffer (6 M guanidine-HCl, 50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 10 mM DTT, pH 8.0). To refold exB4, the denatured exB4 solution was added dropwise to refolding buffer (100 mM Tris-HCl, 2 mM EDTA, 0.4 M L-arginine-HCl, 0.5 mM oxidized glutathione) at 4°C. The refolded exB4 was dialyzed against PBS(-) and used as the immunogen.

[0045] [Example 2] Preparation of anti-human LILRB4 monoclonal antibody Human B4-specific monoclonal antibodies were produced by immunizing mice with the exB4 obtained in Example 1 using the mouse iliac lymph node method (Y. Sado et al., Acta Histochem. Cytochem., 39:89-94, 2006). Next, the monoclonal antibody-containing culture supernatant was screened by ELISA using the following recombinant proteins to select hybridomas producing human B4-specific monoclonal antibodies.

[0046] Recombinant human LILRB1 (Sino Biological Co., Ltd., #16014-H08H) Recombinant human LILRB2 (Sino Biological Co., Ltd., #14132-H08H) Recombinant human LILRB3 (Sino Biological Co., Ltd., #11978-J08H) Recombinant human LILRB4 (Sino Biological Co., Ltd., #16742-H08H) Recombinant human LILRB5 (Sino Biological Co., Ltd., #17221-H08H) Recombinant human LILRA1 (R&D Systems, #9226-T4-050) Recombinant human LILRA2 (R&D Systems, #9040-T4-050) Recombinant human LILRA3 (Sino Biological Co., Ltd., #13549-H08H) Recombinant human LILRA4 (Sino Biological Co., Ltd., #16058-H08H) Recombinant human LILRA5 (R&D Systems, #8956-T4-050) Recombinant human LILRA6 (Sino Biological Co., Ltd., #9088-T4-050) Recombinant mouse LILRB4 (R&D Systems, #9095-T4-050)

[0047] ELISA was performed using F(ab')2 mouse anti-6xHis tag monoclonal antibody (ITM, clone No. 21-48) as the capture antibody and HRP-conjugated rabbit anti-mouse IgG-Fc (ITM) as the detection antibody. The results are shown in Figure 1. In Figure 1, PC indicates the results when antiserum obtained by immunizing mice with human B4 was used instead of the monoclonal antibody-containing culture supernatant, and NC indicates the results when medium was used. As shown in Figure 1, 10-1D (#10), 11-5A (#11), 25-3G (#25), 1-7H-10G-3E (#1), 5-9C-10D-5H (#5), and 31-7G-6H-10H (#31) specifically bound to human B4 but did not bind to other leukocyte Ig-like receptors (LILRs) or mouse B4.

[0048] [Example 3] Inhibition test of B4-B4 ligand binding The ability of the anti-human B4 monoclonal antibody obtained in Example 2 to inhibit the binding of fibronectin to human LILRB4 was examined using B4 chimeric receptor GFP reporter cells. The reporter cells derived from the mouse T cell hybridoma 2B4 express a chimeric receptor consisting of the extracellular domain of human B4 fused to the human activating paired immunoglobulin-like receptor β (PILRβ) and the activating signal adaptor protein DAP12, and contain the green fluorescent protein (GFP) gene, whose expression is induced under the control of the nuclear transcription factor of activated T cells (NFAT) (K Hirayasu et al., Nat. Microbiol. 1:16054, 2016). Using these B4 reporter cells, we examined the inhibitory effect of the anti-B4 monoclonal antibody obtained in Example 2 on the binding of B4 to fibronectin. Before starting the test, a recombinant protein (hereinafter also referred to as FN30-Fc) in which the N-terminal 30 kDa of human fibronectin (Sigma, #9911, hereinafter also referred to as FN30) was fused with the Fc of mouse IgG2a was prepared according to the method described in WO2021 / 029318, and each well of a 96-well flat-bottom plate was precoated with 50 μL of PBS(-) containing 10 μg / mL FN30-Fc at room temperature. Immediately before adding B4 reporter cells, wells were washed three times with PBS(-) for 1 hour. Some wells were left uncoated as negative controls. B4 reporter cells were harvested from the culture and placed on ice until the start of the experiment. 5 × 10 4B4 reporter cells were suspended in 50 μL of hybridoma culture supernatant containing anti-B4 monoclonal antibody and incubated on ice for 30 minutes with gentle mixing every 10 minutes. Next, without washing, the B4 reporter cells were directly added to FN30-Fc-coated or non-coated 96-well plates. Next, 50 μL of medium (RPMI-1640 supplemented with 10% FBS, 50 μM 2-mercaptoethanol, 0.1 U / ml penicillin, and 0.1 μg / ml streptomycin) containing 60 μg / mL recombinant human ApoE (Novoprotein, #CI02) was added to the B4 reporter cells. As a control, 50 μL of medium without human ApoE was added to the B4 reporter cells. The plate was then centrifuged at 400 × g for 3 minutes to attach the B4 reporter cells to the bottom of the plate. B4 reporter cells were incubated at 37°C for 24 hours, and the expression level of GFP protein was measured by flow cytometry. TM Measurements were performed using a FlowJoint III (BD Biosciences). TM Analysis was performed using software (BD Biosciences). The flow cytometry results are shown in Figure 2, the GFP fluorescence intensity in Figure 3, and the geometric mean of the GFP fluorescence intensity in Figure 4. As shown in Figures 3 and 4, anti-human B4 antibodies #10 and #11 were confirmed to effectively inhibit the binding of human B4 to fibronectin and ApoE. It was predicted that #1, #5, #8, and #31 would exhibit inhibitory effects if the antibody concentration in the culture supernatant were increased. Furthermore, since anti-B4 monoclonal antibodies inhibited the binding of B4 to ApoE, it is believed that the B4-binding site on fibronectin and the B4-binding site on ApoE are closely spaced. [Industrial Applicability]

[0049] According to the present invention, there can be provided an anti-human LILRB4 monoclonal antibody or an antibody fragment thereof that specifically binds to human LILRB4 and inhibits the binding of human LILRB4 to FN, an immune checkpoint inhibitor, a therapeutic agent for immune checkpoint-related diseases, and a method for measuring human LILRB4.

Claims

1. An anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or an antibody fragment thereof that specifically binds to the human immunoinhibitory receptor LILRB4 and inhibits the binding of fibronectin to the human immunoinhibitory receptor LILRB4.

2. The anti-human immunoinhibitory receptor LILRB4 monoclonal antibody according to claim 1, wherein the fibronectin binds to the immunoinhibitory receptor LILRB4 via the amino acid sequence represented by SEQ ID NO: 2 in the fibronectin.

3. An immune checkpoint inhibitor comprising the anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or its antibody fragment according to claim 1 or 2 as an active ingredient.

4. A therapeutic agent for immune checkpoint-related diseases, comprising the anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or its antibody fragment according to claim 1 or 2 as an active ingredient.

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

6. The therapeutic agent according to claim 5 , wherein the cancer is a primary cancer.

7. A method for measuring the human immunoinhibitory receptor LILRB4 contained in a biological sample, using the anti-human immunoinhibitory receptor LILRB4 monoclonal antibody or its antibody fragment according to claim 1 or 2.

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