ANTIBODY AGAINST sPLA2-XIIA AND PHARMACEUTICAL COMPOSITION CONTAINING SAME

Abstract

The purpose of the present invention is to provide an antibody against sPLA2-XIIA and a pharmaceutical composition containing the same. The present invention provides: an antibody or an antigen-binding fragment thereof that binds to sPLA2-XIIA, wherein the antibody or antigen-binding fragment thereof inhibits the enzymatic activity of sPLA2-XIIA and inhibits induction of differentiation of naïve T cells into Th17 cells; and a pharmaceutical composition containing the antibody or antigen-binding fragment thereof. The pharmaceutical composition of the present invention can be used, for example, for treating Th17-related diseases or cancer.

Description

Antibody to sPLA2-XIIA and pharmaceutical composition containing same REFERENCE TO RELATED APPLICATIONS

[0001] This application benefits from the priority of an earlier Japanese application, Patent Application No. 2024-85053 (filing date: May 24, 2024), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to sPLA ２ The present invention relates to antibodies against XIIA and pharmaceutical compositions containing the same.

[0003] Phospholipase A ２ (phospholipase A ２ / PLA ２ PLA is a general term for enzymes that hydrolyze the ester bond at the sn-2 position of glycerophospholipids. This enzyme liberates arachidonic acid, a precursor of eicosanoids, from glycerophospholipids, and serves as the starting point for the synthesis of prostaglandins and leukotrienes, which are inflammatory lipid mediators (Non-Patent Document 1). ２ PLA is attracting attention as a therapeutic target for inflammatory diseases. ２ Generally, cPLA is considered to be a ２ (cytosolic PLA ２ ), iPLA ２ (Ca ２＋ -independent PLA ２ ), sPLA ２ (secret PLA ２ It is known that there are a total of more than 50 types of enzymes (isoforms) in these groups due to structural differences. ２ In order to understand the functions of PLA, it is necessary to accurately grasp the lipid metabolism involved in each of them. However, the biological responses involved in these are diverse and intricately intertwined. ２ The biological functions of the

[0004] In recent years, sPLA ２ sPLA, one of the isoforms of ２It has been reported that sPLA-X is involved in the growth of B-cell lymphoma (Non-patent Document 2). ２ -X degrades the membrane phospholipids of tumor cell-derived extracellular vesicles (EVs), resulting in the depletion of sPLA ２ In modified EV, highly unsaturated fatty acids, which are phospholipid degradation products, lysophospholipids, and their metabolites (lipid mediators) accumulate. ２ Macrophages that have taken up modified EVs differentiate into immunosuppressive (i.e., tumor-promoting) tumor-associated macrophages (TAMs), promoting tumorigenesis. ２ Varespladib, an inhibitor of (I, II, V and X), suppresses this response, resulting in the suppression of tumor formation.

[0005] sPLA ２ sPLA, one of the isoforms of ２ The function of -XIIA has long been considered unknown. A comprehensive search for lipid-related genes involved in the differentiation of naive T cells to Th17 cells using CRISPR / Cas9 screening revealed that sPLA- ２ -XIIA was identified (Non-patent Document 3). sPLA secreted by Th17 cells ２ -XIIA generates lysophosphatidylethanolamine (LPE) through lipid metabolism, and LPE acts as a ligand for RORγt, a master transcription factor for Th17 differentiation, and regulates the induction of differentiation into Th17 cells.

[0006] Makoto Murakami, Biochemistry, 90(3), pp. 348-360 (2018) K. Kudo et al., Cell Metab. 34(4), 615-633.e8 (2022) Y. Endo et al., Sci. Immunol. 8(86), eadd4346(2023)

[0007] However, sPLA ２ The inhibitor, varespladib, ２-XIIA. ２ There have been no reports to date on the effects of sPLA-XIIA inhibitors on Th17 cell differentiation induction and tumorigenesis. ２ The involvement of -XIIA in Th17-related diseases and cancer remains unclear.

[0008] The present invention relates to sPLA ２ The present invention aims to provide an antibody against Th17-XIIA and a pharmaceutical composition containing the same. The present invention also aims to provide a pharmaceutical composition for treating Th17-related diseases or cancer. The present invention further aims to provide a method for screening candidate substances for cancer therapeutic agents.

[0009] The present inventors have now discovered that sPLA ２ sPLA inhibits the enzymatic activity of XIIA and suppresses the differentiation of naive T cells into Th17 cells. ２ The present inventors have obtained multiple monoclonal antibodies against sPLA-XIIA. The present inventors have also found that the monoclonal antibodies are effective in treating Th17-related diseases such as psoriasis and rheumatoid arthritis, as well as in the treatment of cancer. ２ The inventors have generated IL-XIIA-deficient animals and found that the IL-XIIA-deficient animals exhibit suppressed Th17 cell differentiation, alleviated symptoms of psoriasis, alleviated symptoms of rheumatoid arthritis, and suppressed tumor formation. The inventors have further found that the obtained monoclonal antibody is useful as a reagent for immunohistochemical staining. The present invention is based on these findings.

[0010] The present invention provides the following: [1] sPLA ２ -An antibody or antigen-binding fragment thereof that binds to sPLA ２[2] The antibody or antigen-binding fragment thereof according to the above-mentioned [1], wherein the antibody is a monoclonal antibody. [3] The antibody or antigen-binding fragment thereof according to [1] or [2] above, wherein the antibody is selected from the group consisting of the following (i), (ii), and (iii): (i) an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4; (ii) an antibody comprising a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 3 with 1 to 12 alterations selected from the group consisting of deletion, substitution, insertion, and addition, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 4 with 1 to 11 alterations selected from the group consisting of deletion, substitution, insertion, and addition; (iii) an antibody comprising a heavy chain variable region consisting of an amino acid sequence having at least 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 3 and a light chain variable region consisting of an amino acid sequence having at least 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 4. [4] A pharmaceutical composition comprising, as an active ingredient, the antibody or antigen-binding fragment thereof according to any of [1] to [3] above. [5] sPLA ２ A pharmaceutical composition for treating a Th17-related disease or cancer, comprising an inhibitor of sPLA-XIIA as an active ingredient, ２A pharmaceutical composition that inhibits the enzymatic activity of β-PLA-XIIA and suppresses the induction of differentiation from naive T cells to Th17 cells. [6] The pharmaceutical composition according to [5] above, wherein the inhibitor is the antibody or antigen-binding fragment thereof according to any of [1] to [3] above. [7] The pharmaceutical composition according to [5] or [6] above, wherein the Th17-related disease is a Th17 cell-dependent autoimmune disease. [8] The pharmaceutical composition according to [7] above, wherein the Th17 cell-dependent autoimmune disease is psoriasis or rheumatoid arthritis. [9] The pharmaceutical composition according to [5] above, wherein the cancer is a solid cancer.

[10] A method for treating a Th17-related disease or cancer, comprising administering to a subject suffering from a Th17-related disease or cancer in need thereof the antibody or antigen-binding fragment thereof according to any of [1] to [3] above, or the pharmaceutical composition according to [4] to [9] above.

[11] sPLA ２

[12] A method for screening candidate substances for cancer therapeutic agents, comprising: (a) selecting a substance that inhibits the enzymatic activity of sPLA-XIIA in the presence of a test substance; ２ (b) quantitating the amount of the enzyme reaction product produced, and selecting the test substance as a candidate substance for a cancer therapeutic agent when the amount produced is lower than the amount produced in the absence of the test substance. ２ The screening method according to the above-mentioned

[12] , wherein the substrate of -XIIA is phosphatidylethanolamine and the enzyme reaction product is lysophosphatidylethanolamine.

[14] sPLA ２

[15] A screening method according to any one of [1] to

[13] above, which comprises selecting a substance that inhibits the enzymatic activity of sPLA-XIIA and suppresses the differentiation of naive T cells into Th17 cells.

[16] A reagent for immunohistochemical staining, comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [3] above as an active ingredient.

[17] A reagent for immunohistochemical staining, comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [3] above as an active ingredient. ２An antibody or antigen-binding fragment thereof that binds to -XIIA, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of (i), (ii), and (iii) below: (i) an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4; (ii) an antibody comprising a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 3 with 1 to 11 alterations selected from the group consisting of deletions, substitutions, insertions, and additions, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 4 with 1 to 11 alterations selected from the group consisting of deletions, substitutions, insertions, and additions; (iii) an antibody comprising a heavy chain variable region consisting of an amino acid sequence that has at least 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 3 and a light chain variable region consisting of an amino acid sequence that has at least 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 4.

[0011] According to the present invention, sPLA ２ The antibody of the present invention is effective in treating diseases involving signal transduction of sPLA-XIIA. ２ -XIIA enzyme activity, in particular sPLA ２ The present invention is advantageous in that it provides a new type of therapeutic agent that inhibits the onset and progression of Th17-related diseases and cancers mediated by -XIIA.

[0012] Figure 1 shows the human and mouse sPLA antibodies produced by 96 hybridomas. ２ 2 shows the results of evaluating the affinity of the antibodies produced by the 30 selected hybridomas for human sPLA-XIIA by ELISA (see Example 1 (1-1)). ２ Figure 3 shows the results of evaluating the inhibitory activity of antibodies produced by 30 selected hybridomas against the enzymatic activity of human sPLA-XIIA (see Example 1 (1-2)). Figure 4 shows the results of evaluating the inhibitory activity of antibodies produced by 30 selected hybridomas against the induction of differentiation into Th17 cells (see Example 1 (1-3)). Figure 5 shows the results of evaluating the inhibitory activity of antibodies produced by 6 selected hybridomas against the enzymatic activity of human sPLA-XIIA (see Example 1 (1-4)). ２FIG. 4B shows the results of evaluating the inhibitory activity against the enzymatic activity of human sPLA-XIIA (see Example 1 (1-4)). FIG. 4B shows the results of evaluating the inhibitory activity against the differentiation induction into Th17 cells for antibodies produced by the six selected hybridomas (see Example 1 (1-4)). The Kruskal-Wallis test was used to test for significance of the inhibitory activity against the differentiation induction into Th17 cells. FIG. 5A shows the results of evaluating the inhibitory activity of monoclonal antibody #44 against the enzymatic activity of human sPLA ２ -XIIA specifically recognizes sPLA ２ Other human sPLA such as -III ２ Figure 5B shows the results of Western blot analysis demonstrating that monoclonal antibody #44 does not recognize human sPLA isoforms (see Example 1 (1-5)). ２ -XIIA and mouse sPLA ２ -XIIA specifically recognizes sPLA ２ -sPLA other than XIIA ２ The results of ELISA show that the anti-sPLA antibody does not recognize the seven isoforms (see Example 1 (1-5)). The significance test for ELISA was performed using the Kruskal-Wallis test. ２ 6A and 6B show the therapeutic effect of anti-sPLA-XIIA monoclonal antibody on psoriasis (see Example 2). Fig. 6A shows the change in ear skin thickness in imiquimod-induced psoriasis model mice when monoclonal antibody #44 and the like were administered. Fig. 6B shows the proportion of Th17 cells in the spleen of imiquimod-induced psoriasis model mice (imiquimod was applied six times) when monoclonal antibody #44 and the like were administered. For significance testing, Sidak's multiple comparisons test with two-way repeated-measures ANOVA was used for Fig. 6A, and Kruskal-Wallis test was used for Fig. 6B. Fig. 7 shows the effect of anti-sPLA-XIIA monoclonal antibody on psoriasis. ２7A and 7B show the therapeutic effect of anti-sPLA-XIIA monoclonal antibody on rheumatoid arthritis (see Example 3). Fig. 7A shows the change in joint swelling in collagen-induced arthritis model mice after administration of monoclonal antibody #44 and the like. Fig. 7B shows the change in clinical score in collagen-induced arthritis model mice after administration of monoclonal antibody #44 and the like. Fig. 7C shows the proportion of Th17 cells in the spleen of collagen-induced arthritis model mice after administration of monoclonal antibody #44 and the like. For significance tests, Sidak's multiple comparisons test with two-way repeated-measures ANOVA was used for Figs. 7A and 7B, and the Kruskal-Wallis test was used for Fig. 7C. Fig. 8 shows the effect of anti-sPLA-XIIA monoclonal antibody on rheumatoid arthritis (see Example 3). Fig. 8A shows the effect of anti-sPLA-XIIA monoclonal antibody on rheumatoid arthritis (see Example 3). Fig. 8B ... ２ Figure 8 shows the therapeutic effect of sPLA-XIIA monoclonal antibody on cancer (see Example 4). Figure 8 shows the change in tumor volume in a mouse subcutaneous tumor model when monoclonal antibody #44 and the like were administered. The significance test was performed using Tukey's multiple comparisons test with two-way ANOVA. Figure 9 shows the therapeutic effect of sPLA-XIIA monoclonal antibody on cancer (see Example 4). ２ 9A is a diagram showing the suppression of Th17 cell differentiation in XIIA-deficient mice compared with wild-type mice (see Example 5 (5-2)). FIG. 9A is a diagram showing the gene expression status of naive T cells (Th0) and Th17 cells in wild-type and XIIA-deficient mice, respectively, by microarray analysis. FIG. 9B is a diagram showing pathways in which gene expression is reduced in T cells of XIIA-deficient mice compared with wild-type mice. FIG. 9C is a diagram showing the results of quantitative PCR measurement of the expression of Th17 markers (Rorc, Il23r, Il17a, and Il17f genes) in naive T cells (Th0) and Th17 cells in wild-type and XIIA-deficient mice, respectively. Tukey's multiple comparisons test with one-way ANOVA was used to test for significance. FIG. 10 is a diagram showing the suppression of Th17 cell differentiation in XIIA-deficient mice compared with wild-type mice, respectively, by microarray analysis. ２10A is a diagram showing the change in ear skin thickness in imiquimod-induced psoriasis model mice for wild-type and XIIA-deficient mice. FIG. 10B is a diagram showing the number of Th17 cells in the spleen and skin of imiquimod-induced psoriasis model mice for wild-type and XIIA-deficient mice. For significance tests, Sidak's multiple comparisons test with two-way ANOVA was used for FIG. 10A, and Tukey's multiple comparisons test with one-way ANOVA was used for FIG. 10B. FIG. 11 shows the suppression of psoriasis in T cell-specific sPLA ２ 11A is a diagram showing the change in ear skin thickness in imiquimod-induced psoriasis model mice for wild-type and XIIA-deficient mice. FIG. 11B is a diagram showing the proportion of Th17 cells in the spleen and skin of imiquimod-induced psoriasis model mice for wild-type and XIIA-deficient mice. Significance tests were performed using Sidak's multiple comparisons test with two-way ANOVA in FIG. 11A and Mann Whitney test in FIG. 11B. FIG. 12 shows the relationship between fibroblast-specific sPLA ２ 12A is a diagram showing the suppression of psoriasis in sPLA-XIIA-deficient mice compared with wild-type mice (see Example 5 (5-3) c). FIG. 12A is a diagram showing the change in ear skin thickening in imiquimod-induced psoriasis model mice for wild-type and sPLA-deficient mice. FIG. 12B is a diagram showing the proportion of Th17 cells in the spleen of imiquimod-induced psoriasis model mice for wild-type and sPLA-deficient mice. Significance tests were performed using two-way repeated-measures ANOVA in FIG. 12A and the Mann Whitney test in FIG. 12B. FIG. 13 shows the suppression of psoriasis in systemic sPLA-XIIA-deficient mice compared with wild-type mice. ２13A is a diagram showing the alleviation of rheumatoid arthritis in collagen-induced arthritis model mice, comparing them with wild-type mice (see Example 5 (5-4)). FIG. 13A is a diagram showing the change in clinical score of collagen-induced arthritis model mice, for wild-type mice and sPLA-XIIA-deficient mice. FIG. 13B is a diagram showing the number of Th17 cells in the spleen of collagen-induced arthritis model mice, for wild-type mice and sPLA-XIIA-deficient mice. FIG. 13C is a diagram showing the condition of bone tissue in the ankle joint of collagen-induced arthritis model mice, for wild-type mice and sPLA-XIIA-deficient mice. Arrows in the photographs indicate bone erosion. For significance tests, Sidak's multiple comparisons test with two-way repeated-measures ANOVA was used for FIG. 13A, and Tukey's multiple comparisons test with one-way ANOVA was used for FIG. 13B. FIG. 14 is a diagram showing the condition of bone tissue in the ankle joint of collagen-induced arthritis model mice, for wild-type mice and sPLA-XIIA-deficient mice. ２ 14A is a graph showing the change in tumor volume in a mouse subcutaneous tumor model for wild-type and XIIA-deficient mice. FIG. 14B is a graph showing the tumor weight in a mouse subcutaneous tumor model at the end of the experiment (14 days after tumor injection) for wild-type and XIIA-deficient mice. For significance tests, Tukey's multiple comparisons test with two-way ANOVA was used in FIG. 14A, and Mann Whitney test was used in FIG. 14B. FIG. 15 shows the change in tumor volume in a mouse subcutaneous tumor model for wild-type and XIIA-deficient mice. ２ 16 shows the expression of leukocyte markers and cytokines in tumor cells of mice with a systemic deficiency of sPLA-XIIA in comparison with wild-type mice (see Example 5 (5-6)). The significance test was performed using an unpaired t-test. ２ Figure 17 shows the expression of macrophage exhaustion markers and checkpoint-related exhaustion markers in tumor cells of sPLA-XIIA systemically deficient mice compared with wild-type mice (see Example 5 (5-6)). Significance was tested using an unpaired t-test. Figure 18 shows the results of flow cytometry. ２Leukocyte (CD45) expression in tumor cells of mice with a systemic deficiency of XIIA ＋ ), cytotoxic T cells (CD3ε ＋ CD8 ＋ ) and cytotoxic T cells expressing the immune checkpoint molecule PD-1 (PD-1 ＋ CD8α ＋ ) in comparison with wild-type mice (see Example 5 (5-6)). Unpaired t-test was used to test for significance. ＋ ) shows a dot plot of the side scatter signal (SSC) against the fluorescently labeled signal of CD45. ２ 18A shows the time course of tumor volume, and FIG. 18B shows the tumor weight 78 days after cell transplantation. The Mann-Whitney test was used for significance testing. FIG. 19 shows the results of immunohistochemical staining using monoclonal antibody #44 (see Example 6). FIG. 19A shows the results of immunohistochemical staining of skin sections from imiquimod-induced psoriasis model mice using monoclonal antibody #44 for wild-type and deficiency mice. FIG. 19B shows the results of immunohistochemical staining of skin sections from human psoriasis vulgaris model mice using monoclonal antibody #44 for wild-type and deficiency mice. １ Figure 20 shows the results of staining with an antibody. ２ 20 shows the results of measuring the reaction products after performing an enzymatic reaction using lysophospholipid-XIIA. In Fig. 20, the amount of lysophospholipid produced by the enzymatic reaction is shown in terms of the amount (nmol) per minute of enzymatic time and per mg of tissue.

[0013] 1. Antibodies As used herein, "antibody" refers to an immunoglobulin, a protein that has a structure in which two heavy chains and two light chains are associated. The heavy chain consists of a heavy chain variable region (VH), heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3). The light chain consists of a light chain variable region (VL) and light chain constant region (CL). The hinge region is located between CH1 and CH2. Among these, the variable region fragment (Fv) consisting of VH and VL is directly involved in antigen binding and is the region that confers diversity to the antibody. The antigen-binding region consisting of VL, CL, VH, and CH1 is called the Fab region, and the region consisting of the hinge region, CH2, and CH3 is called the Fc region.

[0014] The regions of the variable regions that directly contact the antigen are called complementarity determining regions (CDRs), and the regions other than the CDRs that show relatively little variation are called framework regions (FRs). CDRs are also called hypervariable regions, and are sites with particularly high variability in the primary structure, and are present at three separate locations in the primary structure of the polypeptide chains of the heavy and light chain variable regions. Herein, with regard to the complementarity determining regions of an antibody, the heavy chain complementarity determining regions are referred to as CDRH1, CDRH2, and CDRH3 from the N-terminus of the heavy chain amino acid sequence, and the light chain complementarity determining regions are referred to as CDRL1, CDRL2, and CDRL3 from the N-terminus of the light chain amino acid sequence. The framework regions are present at four separate locations in the primary structure of the polypeptide chains of the heavy and light chain variable regions. Herein, the framework regions of the variable regions are designated as FR1, FR2, FR3, and FR4 from the N-terminus of the heavy chain amino acid sequence and the light chain amino acid sequence.

[0015] Antibodies include polyclonal and monoclonal antibodies, with monoclonal antibodies being preferred. Antibodies may be recombinant proteins (recombinant antibodies) and can be produced in animal cells such as Chinese hamster ovary cells (CHO cells). The origin of the antibody is not particularly limited, and examples include antibodies from non-human animals, non-human mammals (e.g., mouse, rat, and camel antibodies), and human antibodies. Antibodies may also be chimeric, humanized, or human. A "chimeric antibody" refers to an antibody in which the heavy chain variable region and light chain variable region are linked to heavy chain constant regions and light chain constant regions of different species, respectively. A "humanized antibody" refers to an antibody in which the corresponding positions in a human antibody are replaced with amino acid sequences characteristic of antibodies of non-human origin. For example, an antibody may have CDRH1-3 and CDRL1-3 of an antibody produced by immunizing a mouse or rat, with all other regions, including the four framework regions of the heavy and light chains, derived from a human antibody. Humanized antibodies are sometimes called CDR-grafted antibodies because they can be obtained by grafting the CDRs of an antibody derived from a non-human animal onto a human antibody. Methods for producing humanized antibodies, including methods for selecting FRs derived from human antibodies, are known, and those skilled in the art can prepare humanized antibodies from antibodies of non-human origin. The term "humanized antibody" may also include human chimeric antibodies. A "human chimeric antibody" is an antibody in which the constant region of a non-human antibody has been substituted with the constant region of a human antibody. In the case of a human chimeric antibody, from the viewpoint of enhancing ADCC activity, for example, the subtype of the human antibody used for the constant region is IgG. １ A "fully human antibody" (human antibody) means an antibody in which both the variable region, consisting of FRs and CDRs, and the constant region are derived from a human antibody. The antibody may also be a multispecific antibody, for example, a bispecific antibody or a trispecific antibody. The antibody may be an isolated antibody or a purified antibody.

[0016] In the present invention, CDRs can be defined using the conventional definitions of CDRs used in the art, such as Kabat, Chothia, AbM, contact, etc. The Kabat definition is based on sequence variation and is the most commonly used (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0017] As used herein, "antigen-binding fragment" refers to a portion of an antibody that maintains its ability to bind to an antigen. The antigen-binding fragment may comprise the heavy chain variable region or the light chain variable region, or both, of the antibody of the present invention. The antigen-binding fragment may be chimerized or humanized. Examples of antigen-binding fragments include Fab, Fab', F(ab') ２ , Fv antibody (variable fragment of antibody), scFv (single chain Fv), diabody, sc(Fv) ２ (Single chain (Fv) ２ Such antibody fragments can be obtained, but are not limited to, by treating the antibody with an enzyme. For example, Fab can be obtained by digesting the antibody with papain. Alternatively, F(ab') can be obtained by digesting the antibody with pepsin. ２ which can be further reduced to obtain Fab'. As used herein, the term "antibody of the present invention" is intended to encompass antigen-binding fragments of the antibody of the present invention.

[0018] The antibodies of the present invention ２ -XIIA-binding antibody. ２ is secretory phospholipase A ２ is an abbreviation for sPLA ２ -XIIA is sPLA ２ The sPLA recognized by the antibody of the present invention is one of the 11 isoforms of ２ -XIIA is a mammalian-derived sPLA ２-XIIA can be used, but preferably human sPLA ２ -XIIA can be used. ２ The amino acid sequence of human sPLA-XIIA and the nucleotide sequence encoding it are registered with the National Center for Biotechnology Information (NCBI) under accession number NP_110448.2. ２ The nucleotide sequence and amino acid sequence encoding XIIA are as set forth in SEQ ID NOs: 1 and 2, respectively. Methods for preparing recombinant proteins in vitro by genetic engineering are well known to those skilled in the art. For example, the human sPLA used in the present invention can be prepared by the method of ２ -XIIA can be obtained by transforming a prokaryotic or eukaryotic host cell with an expression vector incorporating the cDNA in an expressible state, and producing the target protein from the host cell.

[0019] sPLA recognized by the antibody of the present invention ２ -XIIA may also be derived from mammals other than humans, such as mice, rats, monkeys, etc. ２ The sequence information of -XIIA is publicly known, for example, mouse sPLA ２ The sequence information of -XIIA is registered with NCBI under accession number NP_075685.2 and can be used. The preparation method is the same as that for human-derived.

[0020] The antibodies of the present invention ２ It is an antibody that inhibits the enzymatic activity of sPLA-XIIA. ２ The antibody that inhibits the enzymatic activity of sPLA-XIIA is an antibody that inhibits the enzymatic activity of sPLA-XIIA in the presence of the antibody. ２ -XIIA and its enzyme reaction substrate are incubated at about 37°C and a pH of about 7.5, and the amount of enzyme reaction product produced is less than the amount of enzyme reaction product produced in the absence of the antibody. Examples of such antibodies include antibodies that produce 60% or less of the amount of enzyme reaction product produced in the absence of the antibody, and preferably 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less.

[0021] The antibody of the present invention is also an antibody that inhibits the induction of differentiation from naive T cells to Th17 cells. An antibody that "inhibits the induction of differentiation from naive T cells to Th17 cells" refers to an antibody that, when naive T cells are incubated in the presence of the antibody under conditions that induce differentiation from naive T cells to Th17 cells, results in a number or proportion of Th17 cells that is lower than the number or proportion of Th17 cells induced to differentiate in the absence of the antibody. For example, the antibody is an antibody that is 50% or less, preferably 40% or less, 30% or less, 20% or less, or 10% or less, of the number or proportion of Th17 cells induced to differentiate in the absence of the antibody. The conditions for inducing differentiation from naive T cells to Th17 cells can be determined by referring to the conditions described below.

[0022] As used herein, naive T cells are synonymous with naive CD4-positive T cells (i.e., CD4-positive T cells that have emigrated from the thymus and have not been subjected to antigen stimulation). In one embodiment of the present invention, naive CD4-positive T cells (CD4 ＋ ) are CD62L positive (CD62L ＋ Naive CD4-positive T cells can be isolated from the peripheral blood, spleen, etc. of a mammal using an antibody against a cell surface marker and an apparatus such as a cell sorter.

[0023] As used herein, Th17 cells are a subset of CD4-positive T cells and have the ability to produce IL-17A. Conditions for inducing differentiation of naive T cells into Th17 cells include culture conditions commonly used in the field of lymphocyte culture in a medium containing IL-6 and TGF-β (e.g., TGFβ1 or TGFβ3). Non-limiting examples of lymphocyte culture conditions include the following: Culture temperature: about 30 to 40°C (preferably about 37°C), CO ２ Concentration: about 1 to 10% (preferably about 5%), humidity: about 70 to 100% (preferably about 95 to 100%), culture period: about several days (preferably about 3 days). The induction of differentiation into Th17 cells can be evaluated using IL-17A production as an index.

[0024] The antibodies of the present invention also ２That is, the antibody of the present invention can specifically bind to sPLA-XIIA. ２ -XIIA, but sPLA ２ -sPLA other than XIIA ２ The antibody may be an antibody that does not bind to the isoform. ２ -sPLA other than XIIA ２ The phrase "does not bind to isoforms" means that no antigen-antibody reaction is observed in an analytical method utilizing an antigen-antibody reaction, such as Western blotting or ELISA.

[0025] In one aspect, the antibody of the present invention may be an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence substantially identical to that amino acid sequence, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence substantially identical to that amino acid sequence. Non-limiting examples of such antibodies include humanized antibodies.

[0026] In the present invention, heavy chain variable regions consisting of substantially the same amino acid sequence as that of SEQ ID NO: 3 include, for example, those having one or more (e.g., 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) modifications selected from the group consisting of deletion, substitution, insertion, and addition in an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 3. In the present invention, light chain variable regions consisting of substantially the same amino acid sequence as that of SEQ ID NO: 4 include, for example, those having one or more (e.g., 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) modifications selected from the group consisting of deletion, substitution, insertion, and addition in an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 4. The number of amino acids to be modified can be the same as that produced by known methods such as site-directed mutagenesis, or the same as that produced naturally. The modifications can be consecutive or discontinuous in the amino acid sequence. The modifications can also be multiple homogeneous modifications (e.g., multiple substitutions) or multiple heterogeneous modifications (e.g., a combination of one or more deletions and one or more substitutions).

[0027] As described above, there are three CDRs in each of the primary structures of the polypeptide chains of the heavy and light chain variable regions, and modifications may be made only in the FRs by specifying the CDRs and FRs in the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4. The definition of CDR is known in the art, and a person skilled in the art would be able to identify the CDRs and FRs in the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4.

[0028] Among the amino acid modifications, amino acid substitution refers to the replacement of an amino acid residue constituting an amino acid sequence with a different type of amino acid residue. Amino acid substitution may be, for example, conservative substitution. "Conservative substitution" refers to the replacement of one or more amino acids with another amino acid and / or amino acid derivative so as not to substantially alter the function of the protein. In conservative substitution, the substituted amino acid and the substituted amino acid preferably have similar properties and / or functions, for example. Specifically, chemical properties such as hydrophobicity and hydrophilicity indexes, polarity, and charge, or physical properties such as secondary structure, are preferably similar. Amino acids or amino acid derivatives with similar properties and / or functions are known in the art. Examples of nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged amino acids (basic amino acids) include arginine, histidine, and lysine, and examples of negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid.

[0029] Non-limiting examples of the amino acid modifications include one, two, three, four, five, six, or seven amino acid substitutions, more preferably conservative substitutions.

[0030] In the present invention, heavy chain variable regions consisting of substantially the same amino acid sequence include, for example, those having 90% or more sequence identity (preferably 91% or more, 92% or more, 93% or more, 94% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of SEQ ID NO: 3. In the present invention, light chain variable regions consisting of substantially the same amino acid sequence include, for example, those having 90% or more sequence identity (preferably 91% or more, 92% or more, 93% or more, 94% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of SEQ ID NO: 4.

[0031] Here, "identity" refers to the degree of identity when the sequences to be compared are properly aligned, and refers to the percentage of exact amino acid matches between the sequences. Identity is determined by taking into account, for example, the presence of gaps in the sequences and the properties of the amino acids (Wilbur, Natl. Acad. Sci. USA 80, 726-730 (1983)). The alignment can be performed using any algorithm, specifically, homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183, 63-69 (1990)), or Smith-Waterman (Meth. Enzym., 164, 765 (1988)). Furthermore, identity can be calculated using, for example, a known homology search program such as those described above, for example, by using default parameters in the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI). In the present invention, "identity" is used in a sense that includes "homology."

[0032] 2. Antibody Production The antibody of the present invention can be produced by a method well known to those skilled in the art. ２ The monoclonal antibody can be obtained by immunizing an animal with sPLA-XIIA and an adjuvant, collecting and purifying the polyclonal antibody produced in the animal's body. In the present invention, a monoclonal antibody may also be prepared according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)). Specifically, the monoclonal antibody can be prepared by immunizing an animal with sPLA-XIIA and an adjuvant, collecting and purifying the polyclonal antibody produced in the animal's body. ２ An animal is immunized with -XIIA and an adjuvant, B lymphocytes are obtained from the immunized animal, and then fused with myeloma cells to form hybridomas. Hybridomas that produce the desired antibodies are established, and monoclonal antibodies can be obtained.

[0033] sPLA as an antigen ２ -XIIA is sPLA ２ It can be obtained by introducing a cDNA encoding sPLA-XIIA into a host cell by genetic engineering and producing it from the host cell. ２ A vector capable of expressing sPLA-XIIA is prepared, and the vector is introduced into a host cell to express the gene. ２ -XIIA can be purified.

[0034] To select hybridomas producing the desired antibodies, the antibodies produced by the hybridomas are selected from sPLA ２ The antibody can be screened for by evaluating at least two points: that it is an antibody that inhibits the enzymatic activity of T-XIIA, and that it is an antibody that suppresses the differentiation induction of naive T cells into Th17 cells. Whether or not an antibody has these two properties can be evaluated by the procedure described above in 1. Antibodies.

[0035] Examples of antibodies produced from the hybridomas established in this manner include monoclonal antibody #44 prepared in Example 1 below and several other monoclonal antibodies.

[0036] 3. Uses of the Antibody As will be described in the Examples below, the antibody of the present invention can be used to bind sPLA. ２ -XIIA binding activity, sPLA ２ The antibody of the present invention can also inhibit the differentiation induction of naive T cells into Th17 cells. Therefore, the antibody of the present invention can inhibit the function of sPLA ２ The antibody of the present invention is useful as an active ingredient of an inhibitor of a signal for inducing differentiation into Th17 cells in which IL-XIIA is involved. Therefore, the antibody of the present invention is effective in treating Th17-related diseases caused by the induction of differentiation into Th17 cells. That is, the present invention provides a pharmaceutical for treating Th17-related diseases, which comprises the antibody of the present invention as an active ingredient.

[0037] As used herein, the term "Th17-associated disease" refers to a disease caused by the induction of differentiation of naive T cells into Th17 cells. Non-limiting examples of Th17-associated diseases include Th17 cell-dependent autoimmune diseases such as psoriasis, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, and steroid-resistant asthma, as well as chronic inflammatory diseases.

[0038] According to the Examples described below, the antibody of the present invention significantly inhibited tumor formation. Therefore, the antibody of the present invention is effective in treating cancer. That is, according to the present invention, a pharmaceutical for treating cancer comprising the antibody of the present invention as an active ingredient is provided. Non-limiting examples of cancer include solid cancers (e.g., colon cancer, lung cancer, mesothelioma, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, breast cancer, ovarian cancer, testicular tumor, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, brain tumor, retinoblastoma, skin cancer, sarcoma, malignant bone tumor, bladder cancer) and blood cancers (e.g., leukemias such as acute myeloid leukemia and acute lymphocytic leukemia, and multiple myeloma).

[0039] As used herein, the term "treatment" includes therapeutic treatment and prophylactic treatment, and means alleviating or eliminating the cause of a disease, slowing or stopping its progression, alleviating, ameliorating, or eliminating its symptoms, and / or suppressing the worsening of its symptoms in a patient with a disease, and in the case where the disease is "cancer," it also includes suppressing metastasis or recurrence of the cancer.

[0040] 4. Pharmaceuticals Comprising Antibodies as Active Ingredients The present invention provides pharmaceutical compositions comprising the antibodies of the present invention. The pharmaceutical compositions of the present invention may contain, in addition to the antibodies of the present invention, a pharmaceutically acceptable carrier.

[0041] When the antibody of the present invention is administered to a subject, the administration route is not particularly limited as long as a therapeutic effect for a Th17-related disease or cancer is obtained, but parenteral administration (e.g., intravenous administration, topical administration, subcutaneous administration, intraperitoneal administration) is preferred.

[0042] For parenteral administration, an appropriate dosage form can be selected depending on the specific administration route, and examples include injections. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art (e.g., known methods described in the General Provisions for Preparations of the 18th Edition of the Japanese Pharmacopoeia, etc.). Pharmaceutically acceptable carriers include formulation additives such as excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives. The pharmaceutical composition of the present invention may be dissolved or suspended in an appropriate solvent such as water or physiological saline before use.

[0043] The pharmaceutical composition of the present invention may contain an active ingredient other than the antibody of the present invention, or may be used in combination with an active ingredient other than the antibody of the present invention or a pharmaceutical composition containing such an active ingredient. Non-limiting examples of active ingredients other than the antibody of the present invention include antibody preparations effective in the treatment of Th17-associated diseases (e.g., antibody preparations containing an IL-17A antibody, an IL-23 antibody, a TNFα antibody, or an IL-6 antibody as an active ingredient), antibody preparations effective in the treatment of cancer (e.g., antibody preparations containing a PD-1 antibody, a PD-L1 antibody, or a CTLA4 antibody as an active ingredient), steroid preparations (e.g., corticosteroid preparations), and immunosuppressants (e.g., methotrexate, cyclophosphamide, tacrolimus, azathioprine).

[0044] The dosage of the antibody of the present invention can be determined depending on the sex, age, and body weight of the subject, the disease and symptoms to be treated, the dosage form, the route of administration, etc. In the present invention, the dosage of the antibody of the present invention per adult when administered for the purpose of treating a Th17-related disease can be determined, for example, within the range of 0.0001 mg to 1000 mg / kg body weight, but is not limited thereto. Furthermore, in the present invention, the dosage of the antibody of the present invention per adult when administered for the purpose of treating cancer can be determined, for example, within the range of 0.0001 mg to 1000 mg / kg body weight, but is not limited thereto.

[0045] The administration interval of the antibody of the present invention or the pharmaceutical composition of the present invention can be determined depending on the sex, age, and weight of the subject, the disease and symptoms to be treated, the dosage form, the administration route, and the like. For example, the antibody or pharmaceutical composition of the present invention can be administered multiple times (e.g., 2, 3, 4, 5, or 6 times) at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks.

[0046] The subjects to which the antibody of the present invention or the pharmaceutical composition of the present invention is administered can be humans and non-human mammals, and examples of non-human mammals include mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, and monkeys. The subjects to which the antibody of the present invention or the pharmaceutical composition of the present invention is administered are preferably patients suffering from or at risk of a Th17-related disease or cancer.

[0047] 5. Screening Method According to the present invention, sPLA ２ The present invention provides a method for screening for candidate substances for cancer therapeutic agents, which comprises: (a) detecting sPLA-XIIA in the presence of a test substance; ２ The method can include (a) contacting XIIA with its substrate; and (b) quantifying the enzyme reaction product produced, and selecting the test substance as a candidate substance for a cancer therapeutic agent if the amount produced is lower than the amount produced in the absence of the test substance.

[0048] In step (a), sPLA is ２ -XIIA and its substrate are incubated. ２ Contact between -XIIA and its substrate can be carried out in an aqueous solution at 4 to 37°C (preferably 25 to 37°C) and pH 7 to 8 (preferably about pH 7.5). Furthermore, the test substance in the screening method of the present invention is not limited as long as it is a substance that is a candidate for a therapeutic drug, and examples thereof include low molecular weight compounds, antibodies, and antigen-binding fragments thereof, and may also be active ingredients approved as pharmaceuticals.

[0049] sPLA ２ Non-limiting examples of substrates for sPLA-XIIA include phosphatidylethanolamines (e.g., 1-oleoyl-2-arachidonoyl-phosphatidylethanolamine, 1-palmitoyl-2-arachidonoyl-phosphatidylethanolamine) and phosphatidylcholines (e.g., 1-palmitoyl-2-arachidonoyl-phosphatidylcholine). ２ When the substrate of -XIIA is phosphatidylethanolamine (PE), the enzyme reaction product is lysophosphatidylethanolamine (LPE), and sPLA ２ When the substrate of -XIIA is 1-oleoyl-2-arachidonoyl-phosphatidylethanolamine, the enzymatic reaction product is 1-oleoyl-lysophosphatidylethanolamine.

[0050] In step (b), the amount of enzyme reaction product produced in the presence of the test substance is compared with the amount of enzyme reaction product produced in the absence of the test substance, and if the amount of the former is lower than the latter (for example, if the amount of the enzyme reaction product produced is 60% or less of the amount of enzyme reaction product produced in the absence of the test substance, preferably 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less), the test substance can be selected as a candidate substance for a cancer therapeutic agent.

[0051] The screening method of the present invention may further comprise the steps of: (c) performing a treatment for inducing differentiation of naive T cells into Th17 cells in the presence of a test substance; and (d) quantifying the number or ratio of Th17 cells after differentiation induction, and selecting the test substance as a candidate substance for a cancer therapeutic agent when the number or ratio is lower than the number or ratio in the absence of the test substance.

[0052] In step (c), naive T cells are incubated in the presence of a test substance under conditions that induce differentiation of naive T cells into Th17 cells. The conditions for inducing differentiation of naive T cells into Th17 cells can be the conditions described in 1. Antibodies above.

[0053] In step (d), the number or ratio of Th17 cells in the presence of the test substance is compared with the number or ratio of Th17 cells in the absence of the test substance, and if the former number or ratio is lower than the latter (for example, if it is 50% or less of the number or ratio of Th17 cells in the absence of the test substance, preferably 40% or less, 30% or less, 20% or less, or 10% or less), the test substance can be selected as a candidate substance for a cancer therapeutic agent.

[0054] The test substance in step (c) can be a substance selected in step (b) as a candidate substance for a cancer therapeutic agent, but is not limited to this. Steps (c) and (d) may be performed first, and the substance selected in step (d) as a candidate substance for a cancer therapeutic agent may be used as the candidate substance for step (a), and steps (a) and (b) may then be performed.

[0055] The screening method of the present invention may further comprise, after steps (a) and (b), or steps (a) and (b) and steps (c) and (d), a step (e) of administering the substance selected as a candidate substance for a cancer therapeutic agent to a mammal having cancer (e.g., a cancer model animal or a cancer patient). If a significant cancer therapeutic effect of the candidate substance is confirmed in step (e), the candidate substance can be selected as an active ingredient of a cancer therapeutic agent.

[0056] 6. Immunohistochemical staining agent The present invention provides an immunohistochemical staining agent containing the antibody of the present invention as an active ingredient. As described in Example 6 below, when skin affected by psoriasis was immunohistochemically stained with the antibody of the present invention, sPLA, which is one of the causes of psoriasis, was detected. ２ The dermis, where immune cells and fibroblasts expressing sPLA-XIIA are present, was stained, while the epidermis, where these cells are absent, was hardly stained. ２ -It is useful for immunohistochemical staining of XIIA.

[0057] 7. Treatment Method According to another aspect of the present invention, there is provided a method for treating a Th17-related disease or cancer, the method comprising administering an antibody of the present invention or a pharmaceutical composition of the present invention to a subject suffering from a Th17-related disease or cancer in need thereof. The treatment method of the present invention can be carried out in accordance with the description of the antibody of the present invention and the pharmaceutical composition of the present invention (see sections 1 to 4 above).

[0058] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0059] Animal experiments in the examples were conducted in accordance with the University of Tokyo's Animal Experiment Implementation Regulations. Mice were housed in a specific pathogen-free environment with a 12-hour light / dark cycle (light: 8:00-20:00, dark: 20:00-8:00), a room temperature of 23±1°C, and a humidity of 50±10%, and were allowed to freely consume CE-2 feed (CLEA Japan) and ultrafiltered water.

[0060] Cancer cell culture MC38 cells (Karafast) were cultured in high-glucose Dulbecco's modified Eagle's medium (Fujifilm Wako Pure Chemical Industries) containing 10% (v / v) FBS, 1x Penicillin-Streptomycin Solution (Fujifilm Wako Pure Chemical Industries), 1x Non-essential amino acids solution, 1mM sodium pyruvate, and 1x L-glutamine. The cells were maintained at 37°C and 5% CO ２ The cells were cultured under 5% CO₂ and subcultured every 2 to 3 days.

[0061] Prostate cancer cell line TRAMP-C2 cells (ATCC) were cultured in high-glucose Dulbecco's modified Eagle's medium (Fujifilm Wako Pure Chemical Industries) containing 10% (v / v) FBS, 1x Penicillin-Streptomycin Solution (Fujifilm Wako Pure Chemical Industries), 10 nM Dehydroepiandrosterone (Tokyo Chemical Industry), 5 mg / ml Bovine insulin (Sigma-Aldrich), and 1x L-glutamine. The cells were maintained at 37°C and 5% CO ２ The cells were cultured under 5% CO₂ and subcultured every 3 to 4 days.

[0062] Statistical Analysis Data shown in the Examples are expressed as mean ± SEM unless otherwise specified. Differences between two groups in statistical analysis were determined by either paired t-test, unpaired t-test, or Mann-Whitney U-test, depending on the variance. Differences between multiple groups in statistical analysis were determined by one-way analysis of variance (Sidak's multiple comparisons test with one-way ANOVA), two-way analysis of variance (Tukey's multiple comparisons test with two-way ANOVA), or Kruskal-Wallis test. P<0.05 (*), P<0.01 (**), P<0.001 (***), and P<0.0001 (****) were considered statistically significant. GraphPad Prism (GraphPad) was used for statistical analysis.

[0063] Example 1: Mouse anti-human sPLA ２ (1-1) Preparation of antibody-producing hybridomas and screening using binding as an index Human sPLA ２ The full-length cDNA of IgG2a-XIIA (SEQ ID NO: 1) was inserted into the pCAG-Neo mIgG2a-Fc vector (Fujifilm Wako Pure Chemical Industries, Ltd.), and the Fc fusion protein was expressed in CHO cells transformed with the vector. After purifying the Fc fusion protein, sPLA was isolated by the iliac lymph node method. ２-XIIA-deficient mice (see Example 5 (5-1)) were immunized. Spleen cells obtained from the immunized mice were mixed with the myeloma cell line Sp2 / 0-Ag14 (CRL-1581, ATCC) to carry out cell fusion, and immortalized fused cells were selected to produce hybridomas. Production of hybridomas using the iliac lymph node method was outsourced to ITM Corporation.

[0064] Then, recombinant human and mouse sPLA ２ To select clones with high affinity for XIIA, ELISA was performed using the culture supernatant of the hybridoma. ２ Each of the antibodies, XIIA and XIIIA, was immobilized on a microwell plate overnight at 4°C. After blocking with 1% (w / v) BSA buffer, the culture supernatant was added and the primary antigen-antibody reaction was carried out for 1 hour. After washing with 1x PBS-Tween 20, the secondary antigen-antibody reaction was carried out for 30 minutes in the dark with HRP-conjugated rat anti-mouse IgG1 monoclonal antibody (eBioscience). Color development was carried out using EzELISA TMB (ATTO), and the absorbance at 450 nm was measured.

[0065] The results are shown in Figure 1. Of the 96 hybridoma clones, recombinant human or mouse sPLA ２ Thirty hybridoma clones (#3, #6, #11, #13, #15, #24, #25, #29, #33, #35, #36, #38, #39, #44, #45, #49, #50, #52, #59, #60, #63, #64, #65, #73, #80, #81, #87, #90, #94, #96) that produce antibodies reactive with -XIIA were selected and further screening was performed.

[0066] (1-2) sPLA ２ The 30 hybridoma clones obtained in (1-1) above were further screened for inhibition of enzyme activity. ２ The enzyme activity of sPLA-XIIA was measured using a commercially available assay kit (sPLA ２According to the specifications of the assay kit (Item No. 765001, Cayman Chemical), 50 ng of recombinant human sPLA was used. ２ sPLA-XIIA protein (see (1-1) above) was used for the assay. ２ The candidate antibody against sPLA-XIIA was added to a concentration of 10 μg / ml. ２ We screened for antibodies that efficiently inhibit the in vitro enzymatic activity of sPLA-XIIA. ２ This enzyme uses diheptanoylthioPC (1,2-dithio analogue of diheptanoylphosphatidylcholine) as a substrate.

[0067] The results are shown in Figure 2. Of the 30 hybridoma clones, recombinant human sPLA ２ As hybridoma clones producing antibodies that inhibit the enzymatic activity of -XIIA, 15 clones (#36, #38, #39, #44, #45, #49, #59, #63, #64, #65, #73, #80, #90, #94, #96) were selected, which produced approximately 60% or less of the amount of enzymatic reaction product produced in the absence of the antibody (100%) (inhibiting the enzymatic reaction by 40% or more).

[0068] (1-3) Screening of antibodies that inhibit differentiation into Th17 cells The 30 hybridoma clones obtained in (1-1) above were further screened for their ability to inhibit differentiation into Th17 cells. Specifically, 10 μl of hybridoma culture supernatant containing a candidate antibody was added to 100 μl of a culture system for differentiating naive T cells isolated from wild-type mice into Th17 cells, and the mixture was cultured.

[0069] Isolation of naive T cells and differentiation into Th17 cells were performed as follows: Mouse spleens were crushed, and 2 ml of ACK Lysing buffer (Thermo Fisher Scientific-Gibco) was added. After enzymatic digestion at room temperature for 2 minutes, the cells were passed through a 40 μm filter (Greiner Bio-One) and washed with FACS buffer [PBS, 3% (v / v) fetal bovine serum (Hyclone or Biosera), 2 mM EDTA (Dojindo)]. ＋ CD62L ＋ Naive T cells were isolated using the MojoSort™ Mouse CD4 T Cell Isolation Kit (BioLegend) and CD62L MicroBeads, mouse (Miltenyi Biotec) with an autoMACS Pro Cell Separator (Miltenyi Biotec). ＋ CD62L ＋ T cells were precoated overnight at 4°C with 2 μg / ml anti-CD3ε antibody (clone 145-2C11; BioLegend) and 2 μg / ml anti-CD28 antibody (clone 37.51; BioLegend). For differentiation into Th17 cells, the cells were cultured in complete RPMI 1640 medium [RPMI 1640, 10% (v / v) FBS, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 25 mM HEPES, 1% (v / v) non-essential amino acid solution (all Thermo Fisher Scientific-Gibco)] supplemented with 10 ng / ml recombinant mouse IL-1β (BioLegend), 1 ng / ml mouse IL-6 (BioLegend), 10 ng / ml mouse IL-23 (BioLegend), 2 ng / ml human TGF-β (BioLegend), 2 μg / ml anti-mouse IFN-γ (clone XMG1.2; BioLegend), and 2 μg / ml anti-mouse IL-4 (clone 11B11; BioLegend) for 3 days.

[0070] The degree of differentiation induction into Th17 cells was analyzed by flow cytometry using cytokines accumulated in the cells as an index. ＋ IL-17A ＋ The differentiation induction of Th17 cells was analyzed by flow cytometry as follows: After the Th17 differentiation culture, 10 ng / ml Phorbol-12-myristate-13-acetate (Sigma-Aldrich), 500 nM Ionomycin (Sigma-Aldrich), and GolgiPlug™ (1:1000, BD Bioscience) were added to the cells, and the cells were incubated at 37°C and 5% CO ２ The cells were stimulated for 4 hours at 4°C for 4 hours to allow the cytokines produced to accumulate intracellularly. The reaction was stopped by adding FACS buffer, followed by centrifugation at 500 × g for 5 minutes at 4°C. The supernatant was discarded, and TruStain FcX Fc Receptor Blocking Solution (anti-mouse CD16 / 32 antibody; BioLegend) was added to block nonspecific binding to Fc receptors. The cells were incubated on ice for 10 minutes on ice with FACS buffer and centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was discarded, and the cells were incubated on ice for 30 minutes on ice in the dark with fluorescent antibodies (Table 1) to stain the surface antigens. The cells were added with FACS buffer and centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was discarded, and the cells were fixed with Cytofix / Cytoperm (BD Biosciences) for 20 minutes on ice in the dark. After permeabilization with 1x Perm / Wash Buffer (BD Biosciences), the cells were centrifuged at 500 x g for 5 minutes and washed twice, discarding the supernatant. IL-17A antibody or isotype control antibody [Rat IgG2a, κ (clone eBR2a), eBioscience] diluted with 1x Perm / Wash buffer was added, and intracellular staining was performed for 30 minutes on ice in the dark. 1x Perm / Wash buffer was added, and the cells were centrifuged at 500 x g for 5 minutes at 4°C. The supernatant was discarded, and the sample was passed through a 40 μm cell strainer and analyzed using a BD FACSMelody Cell Sorter (BD Biosciences).

[0071]

[0072] The results are shown in Figure 3. Of the 30 hybridoma clones, recombinant human sPLA ２ As hybridoma clones producing antibodies that inhibit the activity of -XIIA to induce differentiation into Th17 cells, 15 clones (#29, #36, #38, #39, #44, #45, #49, #50, #52, #59, #63, #80, #81, #87, #94) were selected, in which the ratio of differentiated Th17 cells was approximately 30% or less of the ratio of Th17 cells in the absence of the antibody (inhibition of differentiation induction by 70% or more).

[0073] (1-4) sPLA ２ Based on the results of both the above screenings (1-2) and (1-3), we screened for antibodies that inhibit the function of sPLA-XIIA and suppress the induction of differentiation into Th17 cells. ２ Hybridoma clones #36, #38, #44, #49, #59, and #80 were selected from the viewpoint of their potent activity in inhibiting the function of sPLA-XIIA and suppressing the differentiation induction into Th17 cells. Purified monoclonal antibodies were prepared from each of these hybridoma clones, and the antibody concentrations were varied in three stages to detect sPLA. ２ The effect of inhibiting the function of -XIIA and the effect of suppressing the induction of differentiation into Th17 cells were evaluated.

[0074] sPLA ２ The effect of inhibiting the function of Th11A was evaluated according to the procedure of (1-2) above, except that the test monoclonal antibody was contacted with the enzyme at a concentration of 10 ng / ml, 100 ng / ml, or 1 μg / ml. The effect of suppressing differentiation induction into Th17 cells was evaluated according to the procedure of (1-3) above, except that the test monoclonal antibody was added at a concentration of 0.5 μg / ml, 1 μg / ml, or 10 μg / ml and cultured.

[0075] The results are shown in Figure 4. Both antibodies inhibited sPLA in a concentration-dependent manner. ２It was confirmed that the monoclonal antibody #44 inhibits the function of sPLA-XIIA and suppresses the differentiation induction into Th17 cells. ２ It showed strong activity in both inhibiting the function of -XIIA and suppressing the induction of differentiation into Th17 cells.

[0076] (1-5) Evaluation of the specificity of monoclonal antibody #44 Next, the specificity of monoclonal antibody #44 was evaluated. Specifically, the specificity of monoclonal antibody #44 was evaluated against human and mouse sPLA. ２ -XIIA and sPLA ２ Human sPLA other than -XIIA ２ Reactivity to the isoforms was assessed by Western blotting and ELISA.

[0077] Western blotting was performed as follows: Polyacrylamide electrophoresis was performed using a 10% gel. The sample consisted of 50 ng of recombinant human and mouse sPLA. ２ -XIIA and 50 ng of human sPLA ２ 5 μl of sample buffer [final concentration: 62.5 mM Tris-HCl, 2% (v / v) SDS, pH 6.8, 25% (v / v) glycerol, 0.01% (v / v) bromophenol blue, 10% (v / v) 2-mercaptoethanol (all Fujifilm Wako Pure Chemical Industries, Ltd.)] was added to 15 μl of each aqueous solution containing ATP-III (control enzyme) and then heated at 95°C for 10 minutes. The gels were placed in an electrophoresis chamber (Rio-Rad) and loaded with the samples. The stacking gel was electrophoresed at 100 V, and the separating gel was electrophoresed at 120 V. The samples were transferred to a PVDF membrane (Merck Millipore) at 108 mA for 30 minutes using a semi-dry rocking apparatus (Bio-Rad). The transfer membrane was blocked for 2 hours using Block Ace (DS Pharma Biomedical). Biotinylated monoclonal antibody #44 or biotinylated anti-human sPLA ２The IgE-III antibody (Kitayama Laboratories) was diluted 1,000-fold with Can Get Signal Solution 1 (Toyobo) and incubated overnight at 4°C. After washing for 5 minutes with 1x TBST [50 mM Tris, 140 mM NaCl, 2.5 mM KCl, 0.005% Tween-20 (all Fujifilm Wako Pure Chemical Industries, Ltd.)], the secondary antibody [HRP streptavidin (BioLegend) or HRP anti-rat IgG (Chemicon)] diluted 1,000-fold with Can Get Signal Solution 2 (Toyobo) was added and incubated at room temperature for 1 hour. After washing five times with 1× TBST for 5 minutes, the sections were incubated with ECL (SuperSignal West Pic PLUS Chemiluminescent Substrate; Thermo Fisher Scientific), and proteins were detected using a chemiluminescence imaging system (FUSION Solo S; Vilber Lourmat).

[0078] Monoclonal antibody #44 and anti-human sPLA ２ Biotin labeling of the Ig-III antibody was performed using a Biotin-Labeling Kit (Dojindo). Specifically, 1 mg / ml of monoclonal antibody #44 was mixed with 1 / 5 the volume of the antibody solution of Reaction buffer and DMSO (Fujifilm Wako Pure Chemical Industries). After mixing with reactive biotin, the mixture was incubated at 37°C for 10 minutes. 28 μl of Stop solution was added, and the mixture was incubated at 37°C for 10 minutes.

[0079] ELISA was performed as follows: 0.5 μg / ml recombinant human or mouse sPLA ２Various isoforms of IgG were immobilized on a microwell plate overnight at 4°C. After blocking with 1% (w / v) BSA buffer, 1 μg / ml of monoclonal antibody #44 was added and a primary antigen-antibody reaction was carried out for 1 hour. After washing with 1x PBS-Tween 20, a secondary antigen-antibody reaction was carried out for 30 minutes in the dark with an HRP-conjugated rat anti-mouse IgG1 monoclonal antibody (eBioscience). Color development was carried out using EzELISA TMB (ATTO), and absorbance was measured at 450 nm.

[0080] The results are shown in Figure 5. Western blotting showed that monoclonal antibody #44 binds to human (h) and mouse (m) sPLA. ２ -XIIA, but showed cross-reactivity to sPLA ２ No cross-reactivity was observed with anti-human sPLA-III (Fig. 5A). ２ -III antibody is human sPLA ２ -III, but showed cross-reactivity with mouse sPLA ２ ELISA showed that monoclonal antibody #44 also showed little cross-reactivity with human sPLA-XIIA (Fig. 5A). ２ -XIIA and mouse sPLA ２ -Antigen-antibody reaction with XIIA was observed, but sPLA ２ -sPLA other than XIIA ２ The antigen-antibody reaction with the seven isoforms was at the same level as the negative control (no primary antibody), and no antigen-antibody reaction was observed (Fig. 5B). ２ It was shown to be an antibody that specifically binds to -XIIA.

[0081] (1-6) Analysis of Variable Region Sequence of Monoclonal Antibody #44 The variable region sequence of monoclonal antibody #44 was analyzed using hybridoma #44 obtained in Example 1. The hybridoma was cultured in IMDM medium (Thermo Fisher Scientific) containing 10% (v / v) FBS, 1x Penicillin-Streptomycin Solution (Fujifilm Wako Pure Chemical Industries, Ltd.), 1x ITS-X (Thermo Fisher Scientific), 1x HT Supplement (Thermo Fisher Scientific), 5% (v / v), BM Condimed H1 (Roche), and 1x L-glutamine. 4.0 x 10 ６ Hybridoma #44 was washed with 20 ml of PBS(-), centrifuged at 200 x g for 5 minutes, and the supernatant was discarded to obtain a cell pellet. This was frozen at -80°C to obtain a frozen pellet. Analysis of the antibody variable region sequence was outsourced to Kazusa Genome Technologies, Inc. The results are shown in Table 2.

[0082]

[0083] Example 2: Anti-sPLA ２ Therapeutic effect of XIIA monoclonal antibody on psoriasis The therapeutic effect on psoriasis was evaluated using imiquimod-induced psoriasis model mice. Specifically, Veselna Cream (Mochida Pharmaceutical) was applied repeatedly to the ear skin of C57BL / 6 male mice (Japan SLC) every day (six times in total) to create imiquimod-induced psoriasis model mice. The model mice were divided into three groups, and IgG １ The mice were divided into three groups: an antibody administration group (n=5), a monoclonal antibody #44 administration group (n=5), and an anti-IL-17A antibody administration group (n=5). The test antibody was intraperitoneally administered to the mice at a dose of 1 mg / kg every day (six times in total) immediately before cream application. IgG １ The antibodies used were GoInVivo™ Purified Mouse IgG1, κ isotype control antibody (BioLegend), and anti-mouse IL-17A antibody (clone 17F3; BioXCell).

[0084] The cream was applied immediately after each measurement of ear thickness using a micrometer (Mitutoyo), and skin thickening was determined. The day the cream was first applied (i.e., the day the ear thickness was first measured) was designated as day 0. However, on day 6, only ear thickness measurements were performed, and no cream was applied. The changes in ear thickness for each group are shown in Figure 6A. Administration of monoclonal antibody #44 to psoriasis model mice significantly inhibited the increase in ear skin thickening, and the effect was comparable to that of anti-IL-17A antibodies used as a therapeutic agent for psoriasis.

[0085] After measuring ear thickness on day 7, spleens were removed from each group of mice and analyzed for CD3ε using flow cytometry. ＋ IL-17A ＋ The number of Th17 cells was evaluated by isolating them. ＋ IL-17A ＋ The proportion of Th17 cells is shown in Figure 6B. When monoclonal antibody #44 was administered to psoriasis model mice, the induction of Th17 cells in the spleen was significantly suppressed, and the effect was comparable to that of anti-IL-17A antibodies used as therapeutic agents for psoriasis. These results demonstrate that the antibodies of the present invention are effective in treating psoriasis.

[0086] Example 3: Anti-sPLA ２ Therapeutic Effect of XIIA Monoclonal Antibody on Rheumatoid Arthritis A mouse model of collagen-induced arthritis was prepared as follows: An equivalent volume of type II collagen (Chondrex) was added dropwise to complete Freund's adjuvant (Chondrex), stirred for 2 minutes, and then allowed to stand on ice for 5 minutes. This procedure was repeated twice, after which the quality of the adjuvant was confirmed. 100 μl of this emulsion was injected subcutaneously into the tails of mice (primary immunization). The start date of primary immunization was designated as day 0. Twenty-one days after primary immunization (day 21), 100 μl of a mixture of incomplete Freund's adjuvant (Chondrex) and type II collagen (Chondrex) prepared by the method described above was injected subcutaneously into the tails of mice (secondary immunization).

[0087] The model mice were divided into three groups: an isotype control antibody administration group (n = 5), a monoclonal antibody #44 administration group (n = 5), and an anti-IL-17A antibody administration group (n = 5). The test antibody was intraperitoneally administered to the mouse arthritis model at a dose of 1 mg / kg two days before primary immunization, and then administered at the same dose and by the same administration route on days 0, 7, 14, 21, 28, 35, and 42.

[0088] After secondary immunization, changes in joint swelling and clinical scores associated with pathology were recorded, and spleen tissue was collected on the final day of the experiment and subjected to cell analysis by flow cytometry. Specifically, hind limb swelling was measured with a micrometer on days 21, 28, 35, and 42 after administration, and the clinical scores of the limbs (0: no inflammation observed in any joint; 1: inflammation observed in one of the joints; 2: inflammation observed in two of the joints; 3: inflammation observed in all joints; 4: inflammation observed in all joints, and the entire leg was red and swollen; a total of 16 points for all limbs) were observed. In addition, spleen tissue was collected from the mice on day 42 and subjected to cell analysis by flow cytometry. Flow cytometry specifically involved CD3ε. ＋ IL-17A ＋ The effect on inducing differentiation into Th17 cells was evaluated using the induction of differentiation into Th17 cells as an index.

[0089] The results are shown in Figure 7. Administration of monoclonal antibody #44 to a collagen-induced arthritis model suppressed joint swelling and clinical scores, and also significantly inhibited the induction of Th17 cells in the spleen (Figures 7A, 7B, and 7C). These effects were comparable to those of anti-IL-17A antibodies used as therapeutic agents for rheumatoid arthritis. These results demonstrate that the antibodies of the present invention are effective in treating rheumatoid arthritis.

[0090] Example 4: Anti-sPLA ２ Therapeutic effect of anti-XIIA monoclonal antibody against cancer ２To investigate the tumorigenesis inhibitory effect of the XIIA monoclonal antibody, a mouse subcutaneous tumor model was performed in which the colon cancer cell line MC38 was transplanted. Specifically, 5.0 × 10 MC38 cells (cultured as described above) were implanted in the pre-shaved abdominal area of ​​C57BL / 6 mice (Japan SLC). ６ The tumors were subcutaneously injected at a volume of 100 μl / 100 μl PBS. From 5 days after administration, an isotype control antibody (n=5) or monoclonal antibody #44 (each dose 1 mg / kg) (n=8) was administered subcutaneously to the tumor site every 3 days. The tumor volume was measured over time using a micrometer from 7 days after administration to 14 days after administration. The tumor volume was calculated using the following formula: Tumor volume (mm ３ ) = 0.5 x (longest diameter) x (shortest diameter) ２

[0091] The results are shown in Figure 8. Administration of monoclonal antibody #44 to a mouse subcutaneous tumor model significantly suppressed tumor formation. These results demonstrate that the antibody of the present invention is effective in cancer treatment.

[0092] Example 5: sPLA ２ (5-1) Creation of knockout animals In this example, sPLA ２ Mice with a total deficiency of sPLA-XIIA and conditional deficiency of sPLA-XIIA were used. ２ Mice with a complete deficiency of sPLA-XIIA (TF0979, heterozygous deficiency + / -) were crossed with each other to generate wild-type + / + mice and homozygous deficiency - / - mice. For the conditional deficiency mice, we first obtained sPLA from The Knockout Mouse Project Repository. ２ A targeting vector for sPLA-XIIA (clone PG00205_Z_2_D12, Project No. 79088) was transfected into mouse embryonic stem cells to establish recombinant embryonic stem cells. Chimeric mice were generated using these recombinant embryonic stem cells by the aggregation method. Chimeric mice (sPLA) in which the mutation was inherited by germline transmission were generated.２ -XIIA flox (Pla2g12a ｆｌ／ｆｌ ) mice) were crossed with T cell-specific Cd4-cre mice (Stock No. 022071; Jackson Laboratory) or fibroblast-specific Col1a2-cre mice (Resource No. nbio228; National Institutes of Biomedical Innovation, Health and Nutrition) to generate CD4 ＋ sPLA in helper T cells or fibroblasts ２ Pla2g12a-XIIA-deficient mice were generated. All experiments were performed using 8- to 12-week-old male mice. C57BL / 6 mice were used in the psoriasis experiment (see (5-3) below), and DBA / 1 mice backcrossed for 10 generations were used in the arthritis experiment (see (5-4) below). As experimental controls, wild-type mice obtained by mating heterozygous deficient mice were used in the case of systemic deficiency, and Pla2g12a-XIIA-deficient mice were used in the case of conditional deficiency. ｆｌ／ｆｌ Mice were used respectively.

[0093] (5-2) Inhibition of Th17 cell differentiation A. RNA extraction and reverse transcription Systemic sPLA ２ Naive T cells were isolated from lymph nodes of XIIA-deficient and wild-type mice and differentiated into Th17 cells ex vivo. Isolation of naive T cells and differentiation and culture of Th17 cells were performed according to the procedures described above (1-3).

[0094] One ml of TRIzol Reagent (Thermo Fisher Scientific-Invitrogen) was added to the differentiated Th17 cells, vortexed, and 1 / 5 of the total volume of chloroform (Fujifilm Wako Pure Chemical Industries) was added. After stirring, the mixture was centrifuged at 20,000 x g at 4°C for 15 minutes, and the supernatant was collected. An equal volume of 2-propanol (Fujifilm Wako Pure Chemical Industries) was added to the supernatant, mixed well, and allowed to stand at -20°C for at least 20 minutes. The mixture was centrifuged at 20,000 x g at 4°C for 15 minutes to precipitate RNA. The supernatant was removed, washed with 150 μL of 70% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries), and centrifuged at 20,000 x g at 4°C for 5 minutes. The supernatant was removed, and the dried precipitate was dissolved in 25 μL of ultrapure water to obtain total RNA. The purity and concentration of the RNA solution were determined by measuring the absorbance at 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). 2 μg of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). 60 μl of ultrapure water was added to this to obtain the cDNA solution.

[0095] (b) Quantitative PCR (qPCR) A PCR reaction system consisting of cDNA, a TaqMan probe / primer set (TaqMan Gene Expression Assay: Il17a, Mm00439618; Il23p19, Mm01160011_g1; Gapdh, 4352339E; Thermo Fisher Scientific or Prime Time qPCR Probes assay: Il17f, Mm.PT.58.9739903.g; Rorc, Mm.PT.58.23464726.g; Integrated DNA Technologies), TaqMan Gene Expression Master Assay Mix (Thermo Fisher Scientific) or PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) was assembled, and qPCR was performed using a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The mRNA expression level was calculated as the ΔCt value (Ct value of the target gene - Ct value of the endogenous control gene Gapdh). -ΔＣｔ was used as the relative gene expression level.

[0096] (c) Microarray analysis and gene ontology analysis. Microarray analysis was performed using Agilent Technologies reagents and equipment. Total RNA was extracted from the naive T cells (Th0 cells) and differentiated Th17 cells prepared in (a) above, and the RNA quality was assayed using a 2100 Bioanalyzer. RNA of sufficient quality was reverse-transcribed and in vitro transcribed for Cy3 labeling using a Low Input QuickAmp Labeling Kit. The prepared cRNA was hybridized overnight with an oligo DNA array containing approximately 44,000 genes (Whole Mouse Genome DNA Microarray Kit, 4x44K). After washing, the array was scanned using a SureScan Microarray Scanner, and the fluorescence intensity of each spot was quantified using Feature Extraction software. GeneSpring GX software was used for normalization of raw data, intergroup comparison of gene expression, and gene ontology (GO) analysis.

[0097] D. Results The results are shown in Figure 9. Microarray analysis revealed that in wild-type T cells, the expression of many genes was induced after differentiation (Th17) compared to before differentiation (Th0), but sPLA ２ This induction was significantly reduced in sPLA-XIIA-deficient T cells (Fig. 9A). ２ The expression of many genes involved in Th17 immune responses was reduced in T cells from -XIIA-deficient mice (Fig. 9B). Furthermore, the expression of representative genes (Rorc, Il23r, Il17a, Il17f) that are indicators of differentiation into Th17 cells was examined by qPCR (n = 6-7). ２ It was confirmed that this was significantly reduced in -XIIA-deficient mice (Fig. 9C).

[0098] (5-3) Inhibition of psoriasis A. Systemic sPLA ２ The inhibitory effect on psoriasis was evaluated using imiquimod-induced psoriasis model mice.２ Veselna Cream (Mochida Pharmaceutical) was applied repeatedly daily (six times in total) to the ear skin of XIIA-deficient mice (n = 9) and wild-type mice (n = 9) to create imiquimod-induced psoriasis model mice. Immediately after each cream application, ear thickness was measured using a micrometer (Mitutoyo) to determine skin thickening. The day on which the cream was first applied (i.e., the day on which ear thickness was first measured) was designated as day 0. However, on day 6, only ear thickness measurement was performed, and no cream was applied. The changes in ear thickness for each group are shown in Figure 10A.

[0099] After measuring ear thickness on day 6, the spleen and ear skin were removed from each group of mice and analyzed for TCRβ expression using flow cytometry. ＋ IL-17A ＋ Th17 cells were isolated and the number of Th17 cells was evaluated. ＋ IL-17A ＋ The proportion of Th17 cells is shown in Figure 10B.

[0100] In an imiquimod-induced psoriasis model, a typical Th17-related disease, systemic sPLA ２ Compared with wild-type mice, the increase in ear skin thickening was significantly suppressed in XIIA-deficient mice. Furthermore, FACS analysis of the spleen and skin revealed that systemic sPLA ２ -XIIA-deficient mice had significantly reduced Th17 cells compared with wild-type mice.

[0101] B T cell-specific sPLA ２ -XIIA-deficient mice T cell-specific sPLA prepared according to the procedure in (5-1) above ２ 11A. The spleen and skin were differentiated into Th17 cells. The changes in ear thickness are shown in Fig. 11A. The CD4 expression levels in the spleen and skin were also significantly higher than those in the wild-type mice. ＋ IL-17A ＋ The proportion of Th17 cells is shown in Figure 11B.

[0102] T cell specific sPLA ２In -XIIA-deficient mice, the increase in ear skin thickening was suppressed compared to wild-type mice. Furthermore, FACS analysis of the spleen and skin revealed that T cell-specific sPLA ２ -XIIA-deficient mice had significantly reduced Th17 cells compared with wild-type mice.

[0103] Fibroblast-specific sPLA ２ -XIIA-deficient mice Fibroblast-specific sPLA prepared according to the procedure in (5-1) above ２ XIIA-deficient mice (n = 3) and wild-type mice (n = 3) were used as imiquimod-induced psoriasis model mice in the same manner as in (5-3)A above, and skin thickness was measured and induction of differentiation into Th17 cells in the spleen was confirmed. The change in ear thickness is shown in Figure 12A. In addition, splenic CD3ε ＋ IL-17A ＋ The proportion of Th17 cells is shown in Figure 12B.

[0104] Fibroblast-specific sPLA ２ In -XIIA-deficient mice, the increase in ear skin thickening was suppressed compared to wild-type mice. Furthermore, FACS analysis of the spleen revealed that fibroblast-specific sPLA ２ -XIIA-deficient mice had significantly reduced Th17 cells compared with wild-type mice.

[0105] D. Discussion Systemic, T cell-specific, and fibroblast-specific sPLA ２ In the case of sPLA-XIIA-deficient mice, even when used as an imiquimod-induced psoriasis model mouse, the increase in skin thickening was significantly suppressed compared to wild-type mice, and the induction of differentiation into Th17 cells in the spleen and skin was significantly suppressed compared to wild-type mice. ２ It has been confirmed that -XIIA is involved in the onset and progression of psoriasis.

[0106] (5-4) Alleviation of rheumatoid arthritis The inhibitory effect on rheumatoid arthritis was evaluated using a collagen-induced arthritis model mouse. Specifically, the systemic sPLA ２-XIIA-deficient mice (n=8) and wild-type mice (n=8) were subjected to primary and secondary immunizations according to the procedure of Example 3.

[0107] After the secondary immunization, changes in clinical scores associated with the pathology were recorded according to the procedure described in Example 3. On the final day of the experiment, spleen tissue was collected and subjected to cell analysis by flow cytometry. The results are shown in Figures 13A and 13B. Furthermore, ankle joints were collected on the final day of the experiment to evaluate the presence or absence of bone destruction due to rheumatoid arthritis. Specifically, the collected ankle bone tissue was immersed overnight in 4% (v / v) paraformaldehyde-phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd.), then decalcified using OSTEOSOFT (Merck Millipore) for 11 days (changed every 3 days), and tissue sections were prepared. The results are shown in Figure 13C.

[0108] In a collagen-induced arthritis model, a typical Th17-related disease, systemic sPLA ２ Compared with wild-type mice, the increase in clinical scores associated with pathology was significantly suppressed in XIIA-deficient mice. ２ The number of Th17 cells was significantly reduced in XIIA-deficient mice compared to wild-type mice. Furthermore, observation of histological sections of bone tissue from the ankle joint revealed that bone destruction occurred in wild-type mice, but systemic sPLA ２ In mice lacking sPLA-XIIA, bone destruction did not occur. ２ It has been confirmed that XIIA is involved in the onset and progression of rheumatoid arthritis.

[0109] (5-5) Inhibition of Tumor Formation (1) The inhibitory effect on tumor formation was evaluated using a mouse subcutaneous tumor model (Syngenic model) in which the colon cancer cell line MC38 was transplanted. Specifically, the systemic sPLA ２ M38 cells (cultured as described above) were inoculated at 5.0 × 10 in the previously shaved area (abdomen) of XIIA-deficient mice (n = 28) and wild-type mice (n = 15). ６The mice were subcutaneously injected at a volume of 100 μl / 100 μl PBS. After subcutaneous injection, the tumor volume was measured over time using a micrometer on days 7, 10, 12, and 14. The tumor volume was calculated using the formula described in Example 4. Furthermore, on day 14 after subcutaneous injection, the mice were euthanized, and the weight of the excised tumor tissue was measured.

[0110] The results are shown in Figure 14. As shown in Figure 14A, sPLA ２ In sPLA-XIIA-deficient mice, tumor growth was suppressed to about half that of wild-type mice. ２ The tumor weight in sPLA-XIIA-deficient mice was about two-thirds that of wild-type mice. ２ -XIIA has been confirmed to be involved in cancer growth.

[0111] (5-6) Investigation of the mechanism of tumor formation suppression The mechanism of tumor formation suppression confirmed in (5-5) above was investigated from an immunological perspective. Specifically, tumor tissues were removed from the mice after a certain period of time had elapsed since tumor cell administration, and immune-related factors and immune-related cells were analyzed. Tumor cells were administered to mice according to the procedure in (5-5) above. Specifically, colon cancer cell line MC38 cells were treated with systemic sPLA ２ 1.0 × 10 to XIIA-deficient mice (n = 4) and wild-type mice (n = 4). ６The tumor tissue (400-500 mg) obtained 14 days later was subcutaneously injected at a volume of 100 μl / 100 μl PBS, and pulverized with scissors. Deficiency mice were prepared according to the procedure in Example 5 (5-1). The tumor tissue was immersed in 2 ml of enzyme solution (RPMI 1640 medium containing 250 μg / ml Liberase™, 2000 units / ml DNase I, 5% (v / v) FBS, and 10 mM HEPES) and digested for 15 minutes at 37°C. The tumor tissue was mechanically suspended and dissociated using a 5 ml syringe equipped with an 18 G needle. Another 1 ml of the above enzyme solution was added, and the tissue was digested for 15 minutes at 37°C. After resuspending and dissociating the tissue using a 5 ml syringe equipped with a 21 G needle, the cell suspension was passed through a 40 μm cell strainer to obtain a cell suspension. After centrifugation at 200 × g for 5 minutes, the supernatant was discarded, and red blood cells were lysed using RBC Lysis Buffer (BioLegend). After washing with 10 ml of PBS(-), the cells were centrifuged at 200 × g for 5 minutes, the supernatant was discarded, and the cells were resuspended in FACS Buffer (0.5% (w / v) BSA, 2 mM EDTA / PBS(-)).

[0112] 0.5 × 10 for qPCR ６ ~1.0 x 10 ６Approximately 100 tumor cells were collected, and RNA was extracted and reverse-transcribed in the same manner as in Example 5 (5-2) to obtain cDNA. TaqMan probe / primer set (Cd3e Mm00599684_g1, Cd8a Mm01182108_m1, Foxp3 Mm00475162_m1, Adgre1 Mm03009946_m1, Itgax Mm00498698_m1, Vegfa Mm1281449_m1, Ifng Mm01168134_m1, Gzmb Mm00442837_m1, Pdcd1 Mm01285676_m1, Cd274 Mm03048248_m1; Thermo Fisher Scientific, Arg1 Mm.PT.58.8651372, Nos2 Mm.PT.58.5680554, Sepp1 Mm.PT.58.5680554). qPCR reactions were performed using the following antibodies: Mm.PT.58.32790488, Lag3 Mm.PT.58.29457571, Havcr2 Mm.PT.58.12200466, Tcf7 Mm.PT.58.13528979; Integrated DNA Technologies).

[0113] For flow cytometry, the cell suspension obtained above was diluted to 1.0 x 10 ６ Cells were collected and analyzed using intracellularly accumulated cytokines as an index according to Example 1 (1-3). Specifically, Fc receptor blocking treatment on the cell surface was performed in the same manner as in (1-3) above, and the fluorescently labeled antibodies in Table 3 were prepared in FACS buffer at the indicated concentrations, and the cells were stained. After reacting with the antibodies on ice in the dark for 30 minutes, the cells were washed with FACS buffer. The cells were suspended in FACS buffer containing 1 μg / ml propidium iodide (PI), passed through a 35 μm cell strainer, and analyzed using a FACSMelody cell sorter.

[0114]

[0115] The analysis results of factors that are indicators of immune cell infiltration among the results of qPCR are shown in Figure 15. ２Tumor cells from mice with a systemic deficiency of sPLA-XIIA showed higher expression of leukocyte markers than those from wild-type mice, indicating greater infiltration of leukocytes, including T cells and macrophages. ２ In tumors from mice with a systemic deficiency of IFN-XIIA, IFN-γ tended to increase compared to tumors from wild-type mice, although this was not significantly different from that in mice. These results suggest that immune cells infiltrated the tumor tissues, resulting in an anti-tumor immune response.

[0116] The results of qPCR analysis of exhaustion markers (tumor cells from systemically deficient mice) are shown in Figure 16. ２ Tumor cells from mice with a systemic deficiency of sPLA-XIIA showed higher expression of immune checkpoint-related exhaustion markers compared to tumor cells from wild-type mice. ２ In tumor cells from mice with a systemic deficiency of -XIIA, T cell exhaustion is expected to be progressing. However, the expression of Tcf7, an indicator of T cell activation, was maintained, indicating that T cells remain activated (i.e., have not reached terminal exhaustion).

[0117] The results of flow cytometry are shown in Figure 17. ２ Tumor cells from mice with a systemic deficiency of XIIA expressed leukocytes (CD45 ＋ ), cytotoxic T cells (CD3ε ＋ CD8 ＋ ) and cytotoxic T cells expressing the immune checkpoint molecule PD-1 (PD-1 ＋ CD8α ＋ ) was the most common.

[0118] From the above, sPLA ２ It was shown that mice with a systemic deficiency of IL-XIIA have an environment in which leukocytes are more likely to infiltrate tumor cells, that the tumor cells have an activated immune system, and that PD-1 levels are high. Therefore, it can be said that the antibody of the present invention may be effective when used in combination with a PD-1 inhibitor or PD-L1 inhibitor.

[0119] (5-7) Inhibition of Tumor Formation (2) We also investigated the inhibitory effect on prostate cancer tumor formation. Specifically, we evaluated the inhibitory effect on tumor formation using a mouse subcutaneous tumor model (Syngenic model) implanted with the prostate cancer cell line TRAMP-C2 cells.

[0120] Specifically, the systemic sPLA produced according to the procedure in (5-1) above ２ 1.0 × 10 TRAMP-C2 cells were injected into the flanks of 5 wild-type mice (n = 5) and 5 mice (n = 5) that had been previously shaved. ６ The cells were subcutaneously injected in an amount of 100 μl of PBS per 100 μl of the cells. After the subcutaneous injection, the tumor volume was measured over time. The tumor volume was calculated using the formula described in Example 4. On the 78th day after the subcutaneous injection of the cells, the mice were euthanized and the weight of the excised tumor was measured.

[0121] The results are shown in Figure 18. As shown in Figure 18, sPLA ２ In the sPLA-XIIA-deficient mice, no tumors were formed even after 78 days. ２ -XIIA has been confirmed to be involved in the growth of cancers other than colon cancer.

[0122] Example 6: sPLA ２ Immunohistological staining with neutralizing monoclonal antibody to sPLA-XIIA. Skin sections from imiquimod-induced psoriasis model mice (Example 2) and human psoriasis vulgaris were stained with monoclonal antibody #44 (Example 1). ２ The expression of sPLA-XIIA was analyzed. Specifically, each skin section was deparaffinized and rehydrated. After antigen retrieval using 0.1% (w / v) Proteinase K (Merck Sigma-Aldrich), the tissue sections were permeabilized with NP-40 and then blocked with Block Ace. The tissue sections were then stained with biotinylated anti-sPLA ２ -XIIA antibody (monoclonal antibody #44) or anti-mouse IgG １Primary antibody reactions were performed overnight at 4°C using antibodies (Invitrogen). After washing with PBST, the sections were incubated with APC Streptavidin (BioLegend) at room temperature for 1 hour. After DAPI staining (VECTASHIELD Mounting with DAPI; Victor Laboratories) and mounting, the fluorescent signals were observed under a microscope. The results of immunofluorescence staining are shown in Figure 19.

[0123] Immunohistological staining of the skin of mouse psoriasis models (wild-type mice and knockout mice) with monoclonal antibody #44 showed that the epidermis of wild-type mice was barely stained, but the dermis (immunocytes, fibroblasts) was stained (Fig. 19A). Neither the epidermis nor the dermis of knockout mice was stained (Fig. 19A). Furthermore, immunohistological staining of the skin of human psoriasis vulgaris showed that, as with the mouse psoriasis model, the epidermis was barely stained, but the dermis was stained (Fig. 19B). Control IgG １ The monoclonal antibody of the present invention did not stain at all when the antibody was used, confirming its specificity and demonstrating its usefulness as a reagent for immunostaining.

[0124] Reference example: sPLA for skin lipids ２ In this example, sPLA ２ It was confirmed that sPLA-XIIA acts on skin-derived phospholipids and uses them as a substrate for the enzymatic reaction. Specifically, the back skin of a C57BL / 6 mouse was removed, and total lipids were extracted from the removed skin tissue according to the Bligh & Dyer method (Bligh, E. G. & Dyer, W. J. Can. J. Biochem. Physiol. 37, 911-917 (1959)). Using this skin-derived total lipid as a substrate, human sPLA-XIIA was synthesized. ２ PLA by -XIIA recombinant enzyme ２ The enzyme reaction was carried out under the following conditions: 10 μM total lipids (substrate) including phospholipids, 100 mM Tris-HCl pH 7.4, 4 mM CaCl ２ 400ng / ml human sPLA ２-XIIA (prepared according to Example 1 (1-1)) Reaction system 250 μl Reaction temperature 37°C Reaction time 30 minutes

[0125] After the reaction was completed, the various lysophospholipids, which were the enzyme reaction products, were quantified by mass spectrometry (n = 3). Measurements were performed using a reversed-phase high-performance liquid chromatography system using a C18 column (Phenomenex) combined with a triple quadrupole mass spectrometer (QTRAP 4000 LC-MS / MS system, Sciex) (see K. Yamamoto et al., Methods Enzymol. 583, 101-117 (2017)). The results are shown in Figure 20. Phospholipase A ２ hydrolyzes the ester bond at the 2nd position (sn-2 position) of the glycerol backbone of phospholipids, releasing fatty acids and lysophospholipids. ２ It was confirmed that human sPLA-XIIA produces lysophospholipids, such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysoplasmalogen (P-LPE). It was also confirmed that these lysophospholipids have fatty acids such as 16:0 (palmitic acid), 18:0 (stearic acid), and 18:1 (oleic acid) bound to the 1st position (sn-1) of the glycerol backbone. ２ It was confirmed that phospholipid-XIIA uses phosphatidylcholine (PC), phosphatidylethanolamine (PE), and plasmalogen (P-PE) as substrates for the enzymatic reaction.

Claims

1. sPLA ２ -XIIA-binding antibody or antigen-binding fragment thereof, ２ An antibody or an antigen-binding fragment thereof that inhibits the enzymatic activity of -XIIA and suppresses the induction of differentiation from naive T cells to Th17 cells.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is selected from the group consisting of the following (i), (ii), and (iii): (i) an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4; (ii) an antibody comprising a heavy chain variable region consisting of an amino acid sequence having 1 to 12 alterations selected from the group consisting of deletions, substitutions, insertions, and additions in the amino acid sequence of SEQ ID NO: 3, and a light chain variable region consisting of an amino acid sequence having 1 to 11 alterations selected from the group consisting of deletions, substitutions, insertions, and additions in the amino acid sequence of SEQ ID NO: 4; (iii) an antibody comprising a heavy chain variable region consisting of an amino acid sequence having at least 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, and a light chain variable region consisting of an amino acid sequence having at least 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

4.

4. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2 as an active ingredient.

5. sPLA ２ A pharmaceutical composition for treating a Th17-related disease or cancer, comprising an inhibitor of sPLA-XIIA as an active ingredient, ２ A pharmaceutical composition that inhibits the enzymatic activity of IL-XIIA and suppresses the induction of differentiation from naive T cells to Th17 cells.

6. The pharmaceutical composition according to claim 5, wherein the inhibitor is an antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. The pharmaceutical composition according to claim 5, wherein the Th17-associated disease is a Th17 cell-dependent autoimmune disease.

8. The pharmaceutical composition according to claim 7, wherein the Th17 cell-dependent autoimmune disease is psoriasis or rheumatoid arthritis.

9. The pharmaceutical composition according to claim 5, wherein the cancer is a solid cancer.

10. A method for treating a Th17-related disease or cancer, comprising administering the antibody or antigen-binding fragment thereof described in claim 1 or the pharmaceutical composition described in claim 5 to a subject suffering from a Th17-related disease or cancer in need thereof.

11. sPLA ２ A method for screening candidate substances for cancer therapeutic agents, comprising selecting a substance that inhibits the enzymatic activity of β-XIIA.

12. (a) sPLA in the presence of the test substance ２ The screening method of claim 11, comprising: (a) contacting -XIIA with its substrate; and (b) quantifying the enzyme reaction product produced, and selecting the test substance as a candidate substance for a cancer therapeutic agent if the amount produced is lower than the amount produced in the absence of the test substance.

13. sPLA ２ The screening method according to claim 12, wherein the substrate of -XIIA is phosphatidylethanolamine and the enzyme reaction product is lysophosphatidylethanolamine.

14. sPLA ２ The screening method according to claim 11 or 12, characterized in that a substance that inhibits the enzymatic activity of -XIIA and suppresses the induction of differentiation from naive T cells to Th17 cells is selected.

15. A reagent for immunohistochemical staining, comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2 as an active ingredient.

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