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

Abstract

Provided are: an immune checkpoint inhibitor comprising, as an active ingredient, a substance that inhibits an interaction between an integrin and an immunosuppressive receptor (hereinafter, referred to as LILR) B3; and a therapeutic agent for immune checkpoint-related diseases, the therapeutic agent comprising, as an active ingredient, a substance that inhibits an interaction between an integrin and an LILRB3.

Description

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

[0001] The present invention relates to immune checkpoint inhibitors and therapeutic agents for immune checkpoint-associated diseases. This application claims priority to international application PCT / JP2024 / 013250 filed March 29, 2024, the contents of which are incorporated herein by reference.

[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] The immune checkpoint inhibitor according to [3], wherein the anti-LILRB3 antibody is a monoclonal antibody or a polyclonal antibody. [5] The derivative of the LILRB3 antibody is a F(ab') of the LILRB3 antibody. ２ , F(ab) ２ , Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides comprising an antibody portion. [6] The immune checkpoint inhibitor according to [1] or [2], wherein the substance that inhibits the interaction between ITG and LILRB3 is an anti-ITGβ1 subunit antibody or a derivative thereof. [7] The immune checkpoint inhibitor according to [6], wherein the anti-ITGβ1 subunit antibody is a monoclonal antibody or a polyclonal antibody. [8] The derivative of the ITGβ1 subunit antibody is an F(ab') of the ITGβ1 subunit antibody. ２ , F(ab) ２, Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides containing an antibody portion. [9] A therapeutic agent for immune checkpoint-related diseases, comprising as an active ingredient a substance that inhibits the interaction between ITG and LILRB3.

[10] The therapeutic agent for immune checkpoint-related diseases according to [9], 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.

[11] The therapeutic agent for immune checkpoint-related diseases according to [9] or

[10] , wherein the interaction between ITG and LILRB3 includes the interaction between the ITG β1 subunit and LILRB3.

[12] The therapeutic agent for immune checkpoint-related diseases according to [9] or

[10] , wherein the substance that inhibits the interaction between ITG and LILRB3 is an anti-LILRB3 antibody or a derivative thereof.

[13] The therapeutic agent for an immune checkpoint-related disease according to

[12] , wherein the anti-LILRB3 antibody is a monoclonal antibody or a polyclonal antibody.

[14] The derivative of the LILRB3 antibody is a F(ab') of the LILRB3 antibody. ２ , F(ab) ２ , Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides comprising an antibody portion.

[15] The therapeutic agent for immune checkpoint-related diseases according to [9] or

[10] , wherein the substance that inhibits the interaction between ITG and LILRB3 is an anti-ITGβ1 subunit antibody or a derivative thereof.

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

[15] , wherein the anti-ITGβ1 subunit antibody is a monoclonal antibody or a polyclonal antibody.

[17] The derivative of the ITGβ1 subunit antibody is an F(ab') of the ITGβ1 subunit antibody. ２ , F(ab) ２ , Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides containing an antibody portion.

[0011] The second embodiment of the present invention also includes the following aspects.

[18] An immune checkpoint inhibitor comprising, as an active ingredient, a substance that inhibits the binding between ITG and LILRB.

[19] The immune checkpoint inhibitor according to

[18] , wherein the LILRB is LILRB2.

[20] The immune checkpoint inhibitor according to

[19] , wherein the binding between ITG and LILRB2 comprises the binding between the ITGβ1 subunit and LILRB2.

[21] The immune checkpoint inhibitor according to

[19] or

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

[22] The immune checkpoint inhibitor according to

[18] , wherein the LILRB is LILRB5.

[23] The immune checkpoint inhibitor according to

[22] , 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.

[24] The immune checkpoint inhibitor according to

[22] or

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

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

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

[25] , 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.

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

[25] or

[26] , wherein the LILRB is LILRB2.

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

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

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

[27] or

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

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

[25] or

[26] , wherein the LILRB is LILRB5.

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

[30] , 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.

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

[30] or

[31] , 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 β1 subunit antibody and the anti-LILRB3 antibody was set at 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, while 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.

[0024] 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.

[0025] FIG. 1 shows the results of Western blotting analysis of FAK and Syk phosphorylation using anti-LILRB3 monoclonal antibody #08, anti-LILRB1 monoclonal antibody, and anti-LILRB5 monoclonal antibody.

[0033] Figure 1 shows the results of quantitative analysis of the results of Western blotting analysis of Syk phosphorylation using anti-LILRB3 monoclonal antibody #08, anti-LILRB1 monoclonal antibody, and anti-LILRB5 monoclonal antibody. Figure 2 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. Figure 3 shows the release of TNF-α or IL-6 from monocytes treated with anti-LILRB2 monoclonal antibody or anti-LILRB3 monoclonal antibody.The upper graph shows the release of TNF-α, and the lower graph shows the release of IL-6. This figure shows the results of the 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. This figure shows the competitive effect of an anti-PIR-A / B monoclonal antibody and an anti-ITG β1 subunit antibody on the mouse macrophage cell line RAW264.7. The left figure shows the results using anti-PIR-A / B D1D2 monoclonal antibody 6C1 or anti-PIR-A / B D5D6 monoclonal antibody 11.3 and an anti-ITG β1 subunit antibody, and the right figure shows the results using anti-PIR-A / B D5D6 monoclonal antibody 10.1 and an anti-ITG β1 subunit antibody. This graph shows a statistical analysis of the interference between anti-PIR-A / B D5D6 monoclonal antibody 11.3 and an anti-ITG β1 subunit antibody in the mouse macrophage cell line RAW264.7. This graph shows a statistical analysis of the interference between anti-PIR-A / B monoclonal antibody and an anti-ITG β2 subunit monoclonal antibody in the mouse macrophage cell line RAW264.7. Figure 1 shows the results of Western blotting analysis of FAK and Syk phosphorylation in mouse peritoneal cells using anti-PIR-A / B monoclonal antibody 11.3. Figure 2 shows a statistical analysis of the results of Western blotting analysis of FAK and Syk phosphorylation in mouse peritoneal cells using anti-PIR-A / B monoclonal antibody 11.3. The left panel shows the results for FAK phosphorylation, and the right panel shows the results for Syk phosphorylation. Figure 3 shows the results of Western blotting analysis of FAK and Syk phosphorylation in mouse peritoneal cells prepared from wild-type mice (WT) and PIR-B-deficient mice (KO) that had adhered to fibronectin-coated culture dishes, after addition of an isotype antibody or anti-PIR-A / B monoclonal antibody 11.3 (hereinafter also referred to as 11.3 antibody). "Isotype" indicates the case where an isotype antibody was added, and "mAb11.3" indicates the case where the 11.3 antibody was added. FIG. 12B shows a quantitative analysis of the results of Western blotting analysis of FAK and Syk phosphorylation using the 11.3 antibody in the mouse peritoneal cells shown in FIG. 12A.The upper graph shows the results of FAK phosphorylation, and the lower graph shows the results of Syk phosphorylation. Peritoneal cells prepared from wild-type mice and adhered to fibronectin-coated culture dishes were fixed with paraformaldehyde, and FRET was observed between a fluorescently labeled isotype antibody or anti-PIR-A / B monoclonal antibody and a fluorescently labeled integrin β1 antibody or isotype antibody. B16F10 melanoma cells were injected into B6 mice, and the 11.3 antibody or PD-1 antibody, or the 11.3 antibody and anti-PD-1 antibody simultaneously, were administered intraperitoneally on days 4, 6, 9, and 12 after injection. The number of cancer cell metastases to the lungs and liver was counted on day 20. The left graph shows the number of cancer metastases in the lungs, and the right graph shows the number of cancer metastases in the liver. LLC-Luc cells were injected into B6 mice, and on days 3, 6, 9, and 12 after injection, 11.3 antibody or anti-PD-1 antibody, or 11.3 antibody and anti-PD-1 antibody, were administered intraperitoneally. IVIS analysis of the metastasis and proliferation status throughout the body was performed on day 20. The left figure shows the number of photon counts per second in the whole-body analysis, the middle figure shows the number of photon counts in the head and neck, and the right figure shows the number of photon counts in the abdomen. LLC-Luc cells were injected into B6 mice, and on days 3, 6, 9, and 12 after injection, 11.3 antibody or anti-PD-1 antibody, or 11.3 antibody and anti-PD-1 antibody, were administered intraperitoneally. IVIS images of the metastasis and proliferation status throughout the body were obtained on day 20. 16B shows the results of Western blotting analysis of FAK and Syk phosphorylation using anti-human integrin β1 monoclonal antibody 4B4 or Ts2 / 16 antibody, HMβ1.1 antibody, or anti-LILRB3 monoclonal antibody #08. 16C shows the results of quantitative analysis of the results of Western blotting analysis of FAK and Syk phosphorylation using anti-human integrin β1 monoclonal antibody 4B4 or Ts2 / 16 antibody, HMβ1.1 antibody, or anti-LILRB3 monoclonal antibody #08 shown in FIG. 16A. The upper row shows the results for FAK phosphorylation, and the lower row shows the results for Syk phosphorylation. 16D shows the results of Western blotting analysis of FAK and Syk phosphorylation using anti-LILRB3 monoclonal antibodies #222821 antibody or #08 antibody in a CD14+ monocyte fraction prepared from human peripheral blood mononuclear cells.This figure shows the reaction of anti-LILRB3 monoclonal antibodies, #222821 antibody and #08 antibody, with which portions of the 16-30mer peptide prepared based on the human LILRB3 amino acid sequence. This figure shows the results of ELISA evaluation of the reactivity of culture supernatants obtained from the wells obtained in Example 14 with the LILRBB3-Fc fusion protein. The wells indicated by hatched frames show wells that showed particularly high signals and the corresponding negative controls in the negative plate. This figure shows the results of flow cytometry analysis of 13 wells (1A1, 1G1, 1D4, 1H4, 2B6, 3G1, 3B6, 4H1, 4H2, 4H6, 4H7, 4C8, and 4A9) obtained in Example 14. PC indicates the positive control #08 antibody, and iso indicates the isotype control mouse IgG1 antibody. This figure shows the phosphorylation of Syk by Western blotting after monocytes were stimulated with the supernatants from 13 wells (1A1, 1G1, 1D4, 1H4, 2B6, 3G1, 3B6, 4H1, 4H2, 4H6, 4H7, 4C8, and 4A9) obtained in Example 14. This figure shows the phosphorylation of Syk by Western blotting after monocytes were stimulated with the culture supernatants from single-cloned hybridomas 1A1 and 3G1. PC indicates the positive control #08 antibody, and iso indicates the isotype control mouse IgG1 antibody. This figure shows the results of flow cytometry analysis of 13 wells (2B11, 2C10, 3B7, 3C9, 3D9, 3E1, 3E4, 3E9, 3H3, 4A7, 4B7, 4H10, and 4H12) obtained in Example 15. PC indicates the positive control Ts2 / 16 antibody, and iso indicates the isotype control mouse IgG1 antibody. Figure 1 shows the phosphorylation of Syk by Western blotting after monocytes were stimulated with the supernatants from 13 wells (2B11, 2C10, 3B7, 3C9, 3D9, 3E1, 3E4, 3E9, 3H3, 4A7, 4B7, 4H10, and 4H12) obtained in Example 15. Figure 1 shows the phosphorylation of Syk by Western blotting after monocytes were stimulated with the culture supernatants from single-cloned hybridomas 2B11 and 3C9.PC indicates the positive control Ts2 / 16 antibody, and iso indicates the isotype control mouse IgG1 antibody. This figure shows the results of flow cytometry of rabbit anti-mouse ITGβ1 peptide antibody. This figure shows the effect of rabbit anti-mouse ITGβ1 peptide antibody in enhancing Syk phosphorylation by Western blotting. This figure shows micrographs showing changes in cell morphology of monocytes treated with anti-LILRB3 antibody #08 or human ITGβ1 subunit antibody Ts2 / 16. This figure shows the longitudinal length of monocytes treated with anti-LILRB3 antibody #08 or human ITGβ1 subunit antibody Ts2 / 16. This figure shows a graph showing the release of TNF-α or IL-6 from CD14+ monocytes treated with anti-LILRB3 antibody #08 or human ITGβ1 subunit antibody Ts2 / 16. This is a heat map comparing the expression levels of selected genes in the isotype control, anti-ITGβ1 antibody Ts2 / 16 stimulation, and anti-LILRB3 antibody #08 stimulation groups. In the color range shown in the upper left, expression levels (expressed in log2) increase toward the right, and decrease toward the left. Expression profile similarity was clustered in the direction of gene and antibody stimulation, and is shown by a dendrogram with bold lines. Clustering was performed using Euclidean Similarity Measure and Ward's method Linkage Rule.

[0014] [Immune Checkpoint Inhibitor] The immune checkpoint inhibitor according to the first embodiment of the present invention comprises, 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, an anti-ITG β1 subunit antibody or a derivative thereof, etc.

[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 this embodiment, the substance that inhibits the interaction between ITG and LILRB3 may be an anti-ITG β1 subunit antibody or a derivative thereof.

[0023] The anti-ITGβ1 subunit antibody may be either a monoclonal or polyclonal antibody as long as it reacts with the ITGβ1 subunit, but a monoclonal antibody is preferably used. The antibody can be produced by the same method as the above-mentioned LILRB3 antibody.

[0024] The derivative of the anti-ITG β1 subunit antibody includes, for example, F(ab') of the anti-ITG β1 subunit antibody. ２ , F(ab) ２ , Fab', Fab, Fv, scFv, variants thereof, fusion proteins or fusion peptides containing an antibody portion, and the like.

[0025] In the immune checkpoint inhibitors of the present invention, the anti-LILRB3 antibody or a derivative thereof and the anti-ITGβ1 subunit antibody 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 via 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The nucleotide sequence and amino acid sequence of the ITGβ1 subunit can be found in the database provided by the National Center for Biotechnology Information (NCBI). For the human (Homo sapiens) ITGβ1 subunit, for example, the Entrez GeneID is 3688 (as of February 9, 2025), and the RefSeq ProteinIDs are NP_391988.1 and NP_596867.1 (corresponding to isoforms 1D and 1A). Examples of the mouse (Mus musculus) ITGβ1 subunit include Gene ID 16412 NP_034708.1 (as of February 11, 2025) and RefSeq Protein ID NP_034708.1, and examples of the rat (Rattus norvegicus) ITGβ1 subunit include Gene ID 24511 (as of February 8, 2025) and RefSeq Protein ID NP_058718.2, and other animals are known to have the ITGβ1 subunit. The ITGβ1 subunits are not limited to the above, and other ITGβ1 subunits are also included in the ITGβ1 subunits of the present invention.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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).

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] In the second embodiment, the pharmaceutically acceptable carriers and additives can be the same as those in the first embodiment.

[0055] 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).

[0056] 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.

[0057] [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 is a substance that has the activity of inhibiting the interaction between ITG and LILRB3, and examples thereof include an anti-LILRB3 antibody or a derivative thereof, an anti-ITG β1 subunit antibody or a derivative thereof, etc. Examples of the anti-LILRB3 antibody or a derivative thereof and the anti-ITG β1 subunit antibody or a derivative thereof include those described above.

[0058] 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.

[0059] In the present invention, the immune checkpoint-associated disease is not particularly limited as long as it is a disease in which the immune checkpoint molecule LILRB3 or LILRB is involved, and examples include autoimmune diseases, cancer, inflammatory diseases, Alzheimer's disease, infectious diseases, and allergic diseases.

[0060] 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.

[0061] 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.

[0062] 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).

[0063] 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).

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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).

[0072] 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.

[0073] 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.

[0074] 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).

[0075] 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.

[0076] [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 environment.

[0077] 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).

[0078] 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 relatively weak, possibly depending on the antibody used. Expression of ITG subunits, β1, β2, β3, β7, β8, α4, α5, αV, and αM, was detected at various levels, as shown in Figure 2B. Fibronectin (FN), which has an N-terminal 30 kDa domain (FN30), binds to 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.

[0079] 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.

[0080] 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:

[0081] 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

[0082] 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.

[0083] [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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] As shown in Figures 7A and 7B, enhancement of Syk phosphorylation was observed with anti-LILRB3 monoclonal antibody as well as 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 Syk phosphorylation. On the other hand, the FAK phosphorylation response to these antibodies was less pronounced compared to Syk phosphorylation. Furthermore, as shown in Figures 7C and 7D, enhancement of Syk phosphorylation was also observed with anti-LILRB5 monoclonal antibody, and the effect was comparable to that of anti-LILRB3 monoclonal antibody #08. Based on the above, it is thought that anti-LILRB2 antibody, anti-LILRB5 antibody, and anti-LILRB3 antibody at least functionally dissociate the direct binding between LILRB2 and the ITGβ1 subunit, the direct binding between LILRB5 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.

[0091] 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.

[0092] Example 9: Verification using mouse PIR-B, a functional homolog of human LILRB3 (1) 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.

[0093] 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 monoclonal antibody interference assays of 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.

[0094] 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.

[0095] [Example 10] Verification using mouse PIR-B, a functional molecule homologous to human LILRB3 (2) The mouse PIR-B gene is considered to correspond to human LILRB3 in terms of sequence similarity, but because its known ligand is an MHC class I molecule, it is thought to correspond to human LILRB1 and LILRB2 in terms of ligand recognition. Furthermore, like the human LILRB group, PIR-B has multiple PIR-A type molecules, which are homologous molecules of the activated form, and the amino acid sequences are also very similar, so antibodies have almost no selectivity between PIR-A and PIR-B. Therefore, even if cells are stimulated with the anti-PIR-A / B domain D5D6 antibody 11.3 antibody or administered to mice, it is impossible to distinguish whether the effect is due to an action on PIR-A or a blockade of PIR-B. Therefore, the following experiment was conducted using PIR-B gene-deficient mice to clarify which receptor is responsible for the effects of the anti-PIR-A / B domain D5D6 antibody 11.3 (hereinafter also referred to as the 11.3 antibody). It is known that the expression level of PIR-A on cells is generally lower than that of PIR-B. Peritoneal cells prepared from wild-type mice (WT) and PIR-B-deficient mice (KO) adhered to fibronectin-coated culture dishes, i.e., a cell population rich in macrophages, were treated with an isotype antibody or the 11.3 antibody, and the increase in FAK phosphorylation and Syk phosphorylation, indicators of integrin activation, was analyzed by Western blot. The results are shown in Figures 12A and 12B. As shown in Figures 12A and 12B, increases in FAK phosphorylation and Syk phosphorylation were observed in KO cells independent of 11.3 antibody stimulation, and no further increases in these phosphorylations were observed with the addition of 11.3 antibody. Therefore, although the 11.3 antibody recognizes both PIR-A and PIR-B, these results demonstrate that the receptor that inhibits FAK phosphorylation and Syk phosphorylation is PIR-B, and that PIR-A is not involved. Based on the above, it is concluded that the homologous molecule responsible for the function of human LILRB3 is mouse PIR-B, not PIR-A, both in terms of its inhibitory function and its function of associating with and regulating integrins.

[0096] Example 11: Verification using mouse PIR-B, a functional homolog of human LILRB3 (3) Next, FRET analysis using the 11.3 antibody and an anti-ITGβ1 subunit antibody was performed to verify whether PIR-B and the integrin β1 subunit are adjacent to or associated with each other. Peritoneal cells prepared from wild-type mice and adhered to a fibronectin-coated culture dish, i.e., a cell population rich in macrophages, were fixed with paraformaldehyde, and FRET was observed between a fluorescently labeled isotype antibody or 11.3 antibody and a fluorescently labeled anti-integrin β1 antibody or isotype antibody. The results are shown in Figure 13. As shown in Figure 13, significant FRET was observed between the 11.3 antibody and the anti-ITGβ1 subunit antibody. These results demonstrated that PIR-B and the integrin β1 subunit are adjacent to each other or associated with each other.

[0097] Example 12: Cancer Therapeutic Effect of Mouse PIR-B Antibody The mouse PIR-B antibody 11.3 antibody was used to examine whether the mouse PIR-B antibody has an inhibitory effect on cancer metastasis and proliferation in tumor-bearing mice transplanted with the melanoma B16F10 strain and the luciferase-expressing lung cancer cell line LLC-Luc. Furthermore, whether the inhibitory effect on cancer metastasis and proliferation is enhanced by combined use with the immune checkpoint inhibitor antibody PD-1 (hereinafter also referred to as anti-PD-1 antibody) was examined as follows.

[0098] First, 2 × 10 B16F10 melanoma cells were injected into each group of six B6 mice. ４ The mice were injected into the fundus venous plexus, and on days 4, 6, 9, and 12 after injection, 200 μg / mouse of the 11.3 antibody or anti-PD-1 antibody, or 200 μg / mouse of the 11.3 antibody and anti-PD-1 antibody, were administered intraperitoneally. On day 20, the mice were euthanized, and the number of cancer cell metastases to the lungs and liver was counted visually. The results are shown in Figure 14. As shown in Figure 14, the number of metastases to the lungs and liver of mice transplanted with melanoma B16F10 tended to decrease when the 11.3 antibody or anti-PD-1 antibody was administered, and was significantly reduced particularly when the anti-PD-1 antibody and the 11.3 antibody were administered simultaneously.

[0099] Next, 5 × 10 LLC-Luc cells were administered to 6 B6 mice per group. ５ The mice were injected into the fundus venous plexus, and on days 3, 6, 9, and 12 after injection, 200 μg / mouse of the 11.3 antibody or anti-PD-1 antibody, or 200 μg / mouse of the 11.3 antibody and the PD-1 antibody, were administered intraperitoneally. On day 20, the metastasis and growth status of the whole body was analyzed by IVIS. The results are shown in Figures 15A(a) to (c) and 15B. Figure 15A(a) shows the number of photon counts per second in the whole-body analysis, (b) shows the number of photon counts in the head and neck, and (c) shows the number of photon counts in the abdomen. Figure 15B shows an IVIS image. As shown in Figures 15A and 15B, the levels of cancer metastasis and growth in mice transplanted with luciferase-expressing mouse lung cancer cells LLC-Luc tended to decrease when the 11.3 antibody or anti-PD-1 antibody was administered, with the exception of one animal in the anti-PD-1 antibody group. The number of abdominal metastases, in particular, was significantly reduced when the 11.3 antibody was administered alone, and when the anti-PD-1 antibody and the 11.3 antibody were administered simultaneously. These results indicate that the mouse PIR-B antibody 11.3 exhibits cancer therapeutic efficacy in vivo, and this efficacy was enhanced by combined use with an anti-PD-1 antibody, an immune checkpoint inhibitor. Therefore, the human LILRB3 antibody can be expected to have similar cancer therapeutic efficacy as the mouse PIR-B antibody, as well as a combined effect with an anti-PD-1 antibody.

[0100] Example 13 Inhibitory Effect of Anti-Human ITGβ1 Subunit Antibodies on the Interaction between LILRB3 and Human ITGβ1 Subunit Commercially available anti-human integrin β1 monoclonal antibodies, 4B4 antibody (Beckman Coulter), Ts2 / 16 antibody (BioLegend), and HMβ1.1 antibody (BioLegend), and a commercially available anti-LILRB3 monoclonal antibody, #08 antibody (SinoBiological), were each added (5 μg / ml, 2.5 μg / ml for 4B4 antibody only) to human peripheral blood-derived CD14+ monocyte fractions adhered to human fibronectin-coated culture dishes, and the cells were incubated for 90 minutes, recovered, and subjected to Western blotting. The results are shown in Figures 16A and 16B. As shown in Figures 16A and 16B, the Ts2 / 16 antibody exhibited an enhancement effect on Syk phosphorylation and FAK phosphorylation equivalent to or greater than that of the positive control #08 antibody. The HMβ1.1 antibody, however, exhibited a weak enhancement effect on Syk phosphorylation and FAK phosphorylation. According to the manufacturer's website, this antibody reacts with human ITG. However, when tested by antibody immunostaining, it showed good binding activity to the mouse ITGβ1 subunit, but staining of the ITGβ1 subunit on human monocytes was weak, suggesting that its binding activity to the human ITGβ1 subunit may be weak. Furthermore, it has been reported that the 4B4 antibody inhibits the binding activity of the ITGβ1 subunit to fibronectin (Takada Y and Puzon W, JBC, 1993;268:17597). For this reason, the enhancement effect on Syk phosphorylation and FAK phosphorylation in this example is thought to be low. Conversely, the Ts2 / 16 antibody is known to enhance cell adhesion function without inhibiting the binding of human ITGβ1 subunit to fibronectin (Takada Y and Puzon W, JBC, 1993;268:17597). This cell adhesion function can be interpreted as being due to its effect of enhancing Syk phosphorylation and FAK phosphorylation. In other words, the Ts2 / 16 antibody was found to enhance ITG signaling by inhibiting the interaction between human ITG and LILRB3, rather than agonistically stimulating ITG signaling.

[0101] Example 14: Preparation of anti-human LILRB3 antibodies Anti-human LILRB3 antibodies were prepared according to the following procedure. First, commercially available anti-human LILRB3 antibodies with a high inhibitory effect on the interaction between LILRB3 and the ITGβ1 subunit were identified as follows. CD14+ monocyte fractions prepared from human peripheral blood mononuclear cells were seeded on fibronectin-coated culture dishes, cultured, and allowed to adhere. After 90 minutes of stimulation with 1 μg / ml of #222821 antibody (manufactured by R&D) and #08 antibody (manufactured by Sino Biological), the cells were lysed, proteins were recovered, and the bands of phosphorylated Syk were detected after SDS-PAGE. The results are shown in Figure 17. As shown in Figure 17, the #08 antibody enhanced Syk phosphorylation to a greater extent than the #222821 antibody.

[0102] Next, we identified which parts of the overlapping 16-30mer peptides created based on the human LILRB3 amino acid sequence the #222821 and #08 antibodies react with by reacting the peptides with the corresponding antibodies on a culture dish, followed by a fluorescently labeled secondary antibody, and observing the mixture under a fluorescent microscope. As a result, as shown in Figure 18, we found that the #222821 antibody reacted with D2 of the D1-D4 domains of LILRB3, and the #08 antibody reacted with the region around domain D4.

[0103] As described above, the epitope of antibody #08, which has a high inhibitory activity against the interaction between human LILRB3 and the human ITGβ1 subunit, was identified. Next, in order to obtain an antibody with inhibitory activity comparable to or greater than that of antibody #08, a human LILRB3 recombinant protein containing this epitope was prepared as an Fc fusion protein and used as an antigen to immunize mice to obtain multiple hybridomas. Specifically, since antibody #08 from SinoBiological showed relatively high inhibitory activity among commercially available human LILRB3 antibodies, a cDNA of Ser219-Thr435 consisting of the amino acid sequence shown in SEQ ID NO: 1, which corresponds to the D3 and D4 regions of the extracellular immunoglobulin-like domains (D1-D4) of human LILRB3 (UniProt AAI04994.1 sequence), was chemically synthesized and inserted into a plasmid for preparing the Fc fusion protein. This was introduced into CHO cells, and the LILRB3-Fc fusion protein was recovered from the culture supernatant and purified using a Protein A column to prepare an immunogen. The resulting LILRB3-Fc fusion protein was diluted to 1.5 mg / mL and mixed with Freund's complete adjuvant at a volume ratio of 1:2 to prepare an emulsion. The antigen amount was approximately 0.049 mg / 100 μL / mouse, and the resulting emulsion was administered to 8-week-old female B6.DBA2F mice. １ Four mice were immunized at the tail base. The boost immunization 17 days later was performed with a 1 mg / mL antigen solution, approximately 0.06 mg / 60 μL per mouse, as a secondary immunization two weeks after the primary immunization. Hybridomas were produced by the standard method using the mouse iliac lymph node method (Y. Sado et al., Acta Histochem Cytochem 39:89-94, 2006). Four days after the boost immunization, iliac lymph nodes were collected, and the lymphocyte count was approximately 0.4 × 10 ８Fusion was performed using mouse SP2 myeloma cells with the PEG method, and the cells were seeded onto four 96-well plates. On day 5, the medium in the wells was removed and fresh HAT selection medium was added. After 10 days, culture supernatants were collected sequentially from wells in which cell growth was confirmed and cryopreserved. Bambanker (CS-02-001, Nippon Genetics) was used for cell preservation. Next, the reactivity of the supernatants to the LILRB3-Fc fusion protein was evaluated by ELISA. Specifically, ELISA analysis was performed using a plate coated with the immunization / screening antigen LILRB3-Fc fusion protein, compared with a plate coated with a screening negative antigen. The results are shown in Figure 19. As a result of ELISA analysis, supernatants from 20 wells with high ELISA signals, i.e., a high S / N ratio, were identified as positive candidates. This supernatant was screened by flow cytometry using human peripheral blood mononuclear cells (hPBMCs) and compared with the peak position of antibody #08, which was a positive control, to further narrow down the positive wells. As a result, 13 wells (1A1, 1G1, 1D4, 1H4, 2B6, 3G1, 3B6, 4H1, 4H2, 4H6, 4H7, 4C8, and 4A9) shown in Figure 20 were selected.

[0104] Next, monocytes were stimulated with the supernatants from these positive wells, and the enhancement of Syk phosphorylation was evaluated by Western blotting. The results are shown in Figure 21. The antibody concentration in the hybridoma supernatant was typically 2-3 μg / mL. Although the effect of the antibodies in the supernatants cannot be simply compared to the effect of a positive control antibody at 5 μg / mL, as shown in Figure 21, they exhibited an enhancement of Syk phosphorylation equal to or greater than that of the #08 antibody used as a positive control. Taking into account the ELISA values ​​shown in Figure 19 and the FACS positive level results shown in Figure 20, three wells, 1A1, 1H4, and 3G1, were selected as having a high likelihood of enhancing Syk phosphorylation. Hybridomas from these wells were cloned using the methylcellulose method to obtain single clones. The culture supernatants were then screened again by flow cytometry as described above to select positive clones and identify them as single clones. The methylcellulose method is a semi-solid cloning method using methylcellulose, which can be performed faster and more efficiently than conventional limiting dilution cloning methods. In this method, each cell is immobilized in a viscous semi-solid medium and grows into an individual monoclonal colony, allowing single clones to be obtained by collecting these colonies. During this single clone acquisition process, well 1H4 stopped growing, so further analysis was discontinued.

[0105] Next, human monocytes were stimulated with the culture supernatant of the single-clone hybridoma obtained above in the same manner as above, and the cells were recovered and subjected to Western blotting to compare the effects of enhancing Syk phosphorylation. The results are shown in Figure 22. As shown in Figure 22, the 1A1 and 3G1 clones were confirmed to be monoclonal antibodies that exhibited effects comparable to or greater than those of the positive control antibody #08.

[0106] Example 15: Preparation of anti-human ITGβ1 subunit mouse antibody Next, an anti-human ITGβ1 subunit mouse antibody with high inhibitory activity against the interaction between ILIRB3 and the ITGβ1 subunit was prepared as follows. The His-tagged protein of Gln21-Asp728 [abcam Recombinant Human Integrin beta 1 protein (His tag) (ab219474)] consisting of the amino acid sequence shown in SEQ ID NO: 2, which corresponds to the extracellular domain of the human ITGβ1 subunit (UniProtP05556 sequence), was used as the immunogen. An emulsion was prepared by mixing the ITGβ1-His solution and Freund's complete adjuvant at a volume ratio of 1:2. The antigen amount was approximately 0.022 mg / 100 μL / mouse, and 8-week-old female B6. DBA2F １ Four mice were immunized at the tail base. A booster immunization was performed 17 days after the primary immunization with a 0.6 mg / mL antigen solution, approximately 0.036 mg / 60 μL per mouse. Hybridomas were produced according to standard procedures using the mouse iliac lymph node method (Y. Sado et al., Acta Histochem Cytochem 39:89-94, 2006). Four days after the booster immunization, iliac lymph nodes were harvested, and the lymphocyte count was approximately 0.8 × 10 ８ Fusion was performed using mouse SP2 myeloma cells and PEG fusion was performed using the PEG method. The cells were seeded onto four 96-well plates. On day 6, the medium in the wells was removed and fresh HAT selection medium was added. From day 9 onwards, culture supernatants were collected sequentially from wells in which cell growth was confirmed and cryopreserved. Bambanker (CS-02-001, Nippon Genetics Co., Ltd.) was used for cell preservation. Supernatants from all 384 wells were screened by flow cytometry using human peripheral blood mononuclear cells (hPBMC), and those with peaks higher than, similar to, or slightly weaker than the peak position of the positive control anti-ITGβ1 subunit monoclonal antibody, Ts2 / 16 antibody, were selected. As a result, 13 wells (2B11, 2C10, 3B7, 3C9, 3D9, 3E1, 3E4, 3E9, 3H3, 4A7, 4B7, 4H10, and 4H12) shown in Figure 23 were selected as positive wells.

[0107] Next, human monocytes were stimulated with the supernatants from the selected wells, and cell extracts were prepared and subjected to Western blotting to assess the degree of enhancement of the phosphorylated Syk band. Cell extracts with high inhibitory activity against the interaction between LILRB3 and the ITGβ1 subunit were selected. Specifically, the antibody concentration in the supernatant was estimated at 2-3 μg / mL. As shown in Figure 24, the positive control human ITGβ1 subunit monoclonal antibody Ts2 / 16 antibody showed comparable enhancement of Syk phosphorylation. Two positive wells, 3C9 and 4H12, showed comparable enhancement, and 2B11, a well with a relatively weak enhancement of Syk phosphorylation despite showing a peak equivalent to that of the Ts2 / 16 antibody in flow cytometry, was selected. The positive wells were then cloned using the methylcellulose method to obtain the single clones 2B11 and 3C9. Monocytes were stimulated with these supernatants, and Western blotting confirmed that these clones exhibited enhancement of Syk phosphorylation comparable to that of the positive control Ts2 / 16 antibody.

[0108] [Example 16] Preparation of rabbit anti-mouse ITGβ1 subunit antibody to examine the cancer therapeutic effect of anti-human ITGβ1 subunit antibody in cancer-bearing model mice. Among human ITGβ1 subunit antibodies, the Ts2 / 16 antibody, which has the activity of inhibiting the interaction between LILRB3 and the human ITGβ1 subunit, has been reported to be an antibody with integrin activation ability, although the mechanism of activation was unknown (Takada Y and Puzon WJ Biol Chem 1993; 268:17597-17601). The epitope of this antibody contains the amino acid sequence 207-218 (NKGEVFNELVGK; SEQ ID NO: 15) of the human ITGβ1 subunit. This amino acid sequence is sandwiched between, but does not overlap with, the amino acid sequences 120-182 and 220-231, which are predicted to be binding sites for the extracellular matrix. It has also been reported that the Ts2 / 16 antibody does not inhibit the binding of the human ITGβ1 subunit to the extracellular matrix (Takada Y and Puzon WJ Biol Chem 1993; 268:17597-17601). Therefore, it was considered desirable to use a peptide containing the above epitope without containing the extracellular matrix-binding site as an immunogen. However, because mouse and rat ITGβ1 subunits are highly homologous, it was determined that it was unlikely that an antibody with good affinity would be obtained by using only the region corresponding to the amino acid sequence 207-218 of the human ITGβ1 subunit as an antigen. Therefore, rabbits were used as the host animal. A 25-mer SPFSYKNVLSLTDRGEFFNELVGQQ (SEQ ID NO: 4) corresponding to the 195-219 amino acid sequence, which contains the mouse amino acid sequence DRGEFFNELVGQ (SEQ ID NO: 3) corresponding to the human 207-218 region but does not contain the 120-182 and 220-231 regions, was chemically synthesized and conjugated to a carrier KLH protein to prepare an immunogen. Six rabbits were immunized with this and an adjuvant, followed by booster immunizations to obtain antisera, and anti-peptide IgG was purified using an affinity column to which the immunogen peptide had been immobilized.Furthermore, macrophage-enriched cell populations adhered to plastic culture dishes prepared from mouse peritoneal cavities were stimulated with 5 μg / mL of antibody. Cells were then harvested and subjected to Western blotting to assess the degree of Syk phosphorylation and FAK phosphorylation. Antibodies with high activity in inhibiting the binding of mouse PIR-B to the human ITGβ1 subunit were selected. The results are shown in Figures 26A and 26B. As shown in Figure 26A, IgG antibodies A to F obtained from six rabbits exhibited binding activity to the ITGβ1 subunit on mouse splenocytes, although antibody D was relatively weak in flow cytometry. The Western blot shown in Figure 26B demonstrated an increase in phosphorylated Syk comparable to that of the positive control PIR-A / B antibody, mAb11.3.

[0109] Example 17 RNAseq Analysis of Human Monocytes Treated with an Antibody That Inhibits the Interaction Between LILRB3 and the Human ITGβ1 Subunit RNAseq analysis of human monocytes treated with an antibody that inhibits the interaction between LILRB3 and the human ITGβ1 subunit comprehensively clarified the gene groups that are activated and suppressed in monocytes by this binding inhibition, as follows: First, the morphological changes of CD14+ monocytes stimulated with anti-LILRB3 antibody and human ITGβ1 subunit antibody were examined as follows. Among the anti-LILRB3 antibody and anti-human ITGβ1 subunit antibody, #08 antibody (manufactured by SinoBiologicals), which is a LILRB3 antibody that inhibits the interaction between LILRB3 and the human ITGβ1 subunit, and Ts2 / 16 antibody (manufactured by BioLegend), an anti-human ITGβ1 subunit antibody, were used to stimulate CD14+ monocytes prepared from human peripheral blood mononuclear cells and seeded on human fibronectin-coated plates at a concentration of 5mμg / mL. Images of the cells taken 24 hours later are shown in Figure 27, and the results of measuring the major axis length of approximately 300 cells are shown in Figure 28. As shown in Figures 27 and 28, both antibody stimulations resulted in statistically significant elongation and enlargement (blast formation) compared to isotype antibody stimulation. Furthermore, although this may vary depending on the combination of antibody clones used, there was also a significant difference between the anti-human ITGβ1 subunit antibody and the anti-LILRB3 antibody used. Next, the supernatant obtained at this time was used to measure the TNF-α and IL-6 released in response to stimulation with each antibody using ELISA. The results are shown in Figure 29. As shown in Figure 29, TNF-α was increased by the anti-LILRB3 antibody, and IL-6 was increased in response to stimulation with both the anti-human ITGβ1 subunit antibody and the anti-LILRB3 antibody.

[0110] Next, 24 hours after culturing the antibody-stimulated monocytes, RNA was extracted and subjected to transcriptome analysis (RNAseq analysis). RNAseq was performed as follows. 24 hours after stimulating and culturing with 5 μg / mL of antibody, the culture supernatant was removed, washed, and the cells were lysed with QIAzol to prepare total RNA. Poly(A) + RNA was purified using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), a library preparation pretreatment reagent for NEBNext next-generation sequencers, and then reverse-transcribed to cDNA. The cDNA was then comprehensively analyzed for gene transcription after antibody stimulation using a 50 bp paired-end Illumina NextSeq next-generation sequencer. A sample stimulated with an isotype antibody (mouse IgG) was used as a negative control, and similarly manipulated. From the obtained comprehensive transcript list, gene ontology analysis (GO analysis) was performed to determine which gene groups among the differentially expressed genes had characteristic response pathways. The results of stimulation with an anti-human ITGβ1 subunit antibody (ITGβ1 / iso) versus an isotype antibody, stimulation with an anti-LILRB3 antibody (LILRB3 / iso), and stimulation with an anti-human ITGβ1 subunit antibody versus a LILRB3 antibody (LILRB3 / ITGβ1) were statistically compared. The results are shown in Table 1.

[0111]

[0112] As shown in Table 1, in ITGβ1 / iso and LILRB3 / iso, P<0.001 was observed for most pathways, indicating that gene groups related to innate immune responses and inflammatory responses to viruses and bacteria were significantly activated, and LILRB3 / ITGβ1 significantly increased the level of transcription of genes related to inflammatory responses and cytokine responses. Conversely, inactivated gene groups were suppressed in several metabolic pathways, for example, the gene group for the fatty acid synthesis pathway (fatty acid biosynthesis). This suggests that the cellular state has generally shifted from energy storage to energy production and utilization, i.e., toward an immune response.

[0113] Furthermore, several genes were selected, including macrophage M1 markers (CD80), M2 markers (CLEC10A, PPARGC1B), myeloid-derived suppressor cell markers (CD33, CD11b), cytokines (IL1A, IL1B, IL6, IL10, IL12B, IL23A), Fc receptors (FCER1A), and chemokines (CXCL1, CXCL5, CXCL8, CXCL10, CXCL11), and a heat map was created comparing the extent to which their transcription levels increased or decreased following stimulation with anti-human ITGβ1 subunit antibody or anti-LILRB3 antibody compared to the isotype-stimulated group. The results are shown in Figure 30.

[0114] As shown in Figure 30, monocytes stimulated with anti-LILRB3 antibodies showed a significant increase in M1 markers, while monocytes stimulated with human ITGβ1 subunit antibodies showed an increase in M1 markers, a decrease in M2 markers, and a tendency toward a decrease in MDSC markers. These results, similar to pathway analysis, indicate that gene groups related to innate immune responses and inflammatory responses were significantly activated, and that monocytes were differentiated into M1 macrophages.

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. The immune checkpoint inhibitor of claim 3, wherein the anti-LILRB3 antibody is a monoclonal antibody or a polyclonal antibody.

5. The derivative of the LILRB3 antibody is an F(ab') of the LILRB3 antibody. ２ , F(ab) ２ 5. The immune checkpoint inhibitor according to claim 3 or 4, which is selected from Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides comprising an antibody portion.

6. The immune checkpoint inhibitor according to claim 1 or 2, wherein the substance that inhibits the interaction between the integrin (hereinafter also referred to as ITG) and LILRB3 is an anti-ITGβ1 subunit antibody or a derivative thereof.

7. The immune checkpoint inhibitor of claim 6, wherein the anti-ITG β1 subunit antibody is a monoclonal antibody or a polyclonal antibody.

8. The derivative of the ITGβ1 subunit antibody is an F(ab') of the ITGβ1 subunit antibody. ２ , F(ab) ２ 8. The immune checkpoint inhibitor according to claim 6 or 7, which is selected from Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides comprising an antibody portion.

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

10. The therapeutic agent for an immune checkpoint-associated disease according to claim 9, 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.

11. The therapeutic agent for an immune checkpoint-associated disease according to claim 9 or 10, wherein the interaction between the integrin and LILRB3 includes the interaction between the integrin β1 subunit and LILRB3.

12. The therapeutic agent for immune checkpoint-related diseases according to claim 9 or 10, wherein the substance that inhibits the interaction between integrin and LILRB3 is an anti-LILRB3 antibody or a derivative thereof.

13. The therapeutic agent for an immune checkpoint-associated disease according to claim 12, wherein the anti-LILRB3 antibody is a monoclonal antibody or a polyclonal antibody.

14. The derivative of the LILRB3 antibody is an F(ab') of the LILRB3 antibody. ２ , F(ab) ２ 14. The therapeutic agent for an immune checkpoint-related disease according to claim 12 or 13, which is selected from Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides comprising an antibody portion.

15. A therapeutic agent for immune checkpoint-related diseases according to claim 9 or 10, wherein the substance that inhibits the interaction between the integrin (hereinafter also referred to as ITG) and LILRB3 is an anti-ITGβ1 subunit antibody or a derivative thereof.

16. The therapeutic agent for immune checkpoint-related diseases according to claim 15, wherein the anti-ITGβ1 subunit antibody is a monoclonal antibody or a polyclonal antibody.

17. The derivative of the ITG β1 subunit antibody is an F(ab') of the ITG β1 subunit antibody. ２ , F(ab) ２ 17. The therapeutic agent for an immune checkpoint-related disease according to claim 15 or 16, which is selected from Fab', Fab, Fv, scFv, variants thereof, and fusion proteins or fusion peptides comprising an antibody portion.

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