Diagnosis of immune-mediated inflammatory diseases using MMP12 as indicator, and medicine for treating immune-mediated inflammatory diseases via MMP12 inhibition

Abstract

Provided are: a method for detecting immune-mediated inflammatory diseases characterized by an increase in the expression of MMP12 in a subject; a diagnostic drug containing a substance that specifically interacts with MMP12; and a therapeutic agent containing an MMP12-inhibiting substance.

Description

Diagnosis of immune-mediated inflammatory diseases using MMP12 as an indicator, and medicine for treating immune-mediated inflammatory diseases by inhibiting MMP12

[0001] The present invention relates to a method for detecting immune-mediated inflammatory diseases, a biomarker for diagnosing immune-mediated inflammatory diseases, a diagnostic agent for immune-mediated inflammatory diseases, a therapeutic agent for immune-mediated inflammatory diseases, and a diagnostic kit.

[0002] Granulomatous vasculitis is a type of immune-mediated inflammatory disease, and Takayasu arteritis (TAK), giant cell arteritis (GCA), eosinophilic granulomatosis with polyangiitis (EGPA), granulomatosis with polyangiitis (GPA), which are classified as ANCA (anti-neutrophil cytoplasmic antibody)-associated vasculitis, and microscopic polyangiitis (MPA) are all designated intractable diseases. According to 2020 statistics from the Rare Disease Medical Research Foundation / Rare Disease Information Center, the number of recipient certificate holders is approximately 4,700 patients with Takayasu's arteritis, approximately 1,700 patients with giant cell arteritis, approximately 5,200 patients with eosinophilic granulomatosis with polyangiitis, approximately 3,200 patients with granulomatosis with polyangiitis, and approximately 11,000 patients with microscopic polyangiitis.

[0003] In these granulomatous vasculitis, chronic granulomatous inflammation of the vascular wall leads to vascular occlusion and / or rupture, and the prognosis is generally poor. Disease progression is assessed by imaging diagnostics and elevated serum CRP levels.

[0004] Treatments include immunosuppressive therapies such as steroids (prednisolone (registered trademark)), cyclophosphamide (Endoxan (registered trademark)), and methotrexate (Rheumatrex (registered trademark)), as well as the B-cell targeted therapy rituximab (Rituxan (registered trademark)), the complement C5a selective inhibitor avacopan (Taboones (registered trademark)), and the interleukin-6 (IL-6) inhibitor tocilizumab (Actemra (registered trademark)) (Non-Patent Documents 1 and 2), but long-term administration can cause asymptomatic progression of large blood vessel stenosis and dilation.

[0005] N Engl J Med 2017; 377:317-328Ann Rheum Dis. 2018 Mar;77(3):348-354

[0006] The problem to be solved by the present invention is to provide a method for detecting immune-mediated inflammatory diseases characterized by increased expression of MMP12, which does not use IL-6 as an indicator, a biomarker for diagnosing such immune-mediated inflammatory diseases, a diagnostic agent for such immune-mediated inflammatory diseases, a diagnostic kit for such immune-mediated inflammatory diseases, and a therapeutic agent for such immune-mediated inflammatory diseases.

[0007] The present invention includes the following embodiments: Item 1. A method for detecting an immune-mediated inflammatory disease characterized by increased MMP12 expression in a subject, the method comprising measuring the level of MMP12 protein in a biological sample obtained from the subject. Item 2. The method of Item 1, further comprising contacting the biological sample obtained from the subject with a substance that binds to MMP12, wherein measuring the level of MMP12 protein comprises measuring the level of a complex between MMP12 protein and a substance that binds to MMP12 protein. Item 3. The method of Item 1, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is selected from the group consisting of vasculitic syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis. Item 4. The method of Item 1, wherein the biological sample is blood, serum, or plasma. Item 5. The method of Item 1, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is Takayasu's arteritis. Item 6. The method of Item 1, further comprising contacting the biological sample obtained from the subject with a peptide having a site cleavable by MMP12, and measuring the MMP12 protein level comprises measuring a change caused by cleavage of the peptide having a site cleavable by MMP12. Item 7. The method of any one of Items 1 to 5, wherein the method is for predicting the likelihood of developing an immune-mediated inflammatory disease in a subject characterized by increased MMP12 expression, and further comprises comparing the MMP12 protein level in the biological sample obtained from the subject with a reference value, and a higher MMP12 protein level in the biological sample obtained from the subject compared to the reference value indicates a high likelihood that the subject will develop an immune-mediated inflammatory disease.Item 8. The method according to any one of Items 1 to 5, wherein the method is for predicting the prognosis of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the subject has been treated for the immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the level of MMP12 protein in a biological sample obtained from the subject with a reference value, wherein a higher level of MMP12 protein in the biological sample obtained from the subject compared to the reference value indicates a high possibility of relapse of the immune-mediated inflammatory disease characterized by increased expression of MMP12 in the subject. Item 9. The method according to any one of Items 1 to 5, wherein the method is for predicting a post-treatment state of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the subject has been treated for the immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the MMP12 protein level in a biological sample obtained from the subject with a reference value, wherein a lower MMP12 protein level in the biological sample obtained from the subject compared to the reference value indicates a high likelihood that the immune-mediated inflammatory disease characterized by increased expression of MMP12 in the subject is in remission. Item 11. The method according to any one of Items 1 to 5, wherein the method is for evaluating the timing of administration of a therapeutic agent for an immune-mediated inflammatory disease characterized by increased MMP12 expression to a subject suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression, and the method further comprises comparing the MMP12 protein level in a biological sample obtained from the subject with a reference value, and a lower MMP12 protein level in the biological sample obtained from the subject compared to the reference value indicates that administration of the agent to the subject should be discontinued. Item 12. A biomarker for diagnosing an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising MMP12. Item 13. A diagnostic agent for an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising a substance that specifically interacts with MMP12.Item 13. The diagnostic agent according to Item 12, which comprises a conjugate of a peptide having a site cleavable by MMP12 and a labeling substance. Item 14. The diagnostic agent according to Item 12, which comprises a conjugate comprising a peptide having a site cleavable by MMP12, a fluorophore attached to one end of the peptide, and a quencher attached to the other end of the peptide. Item 15. The diagnostic agent according to any of Items 12 to 14, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis. Item 16. A diagnostic kit for diagnosing an immune-mediated inflammatory disease characterized by increased MMP12 expression, which comprises a substance that specifically interacts with MMP12 protein. Item 17. Item 17. The diagnostic kit according to Item 16, wherein the substance that specifically interacts with MMP12 protein comprises an antibody against MMP12, an antigen-binding fragment of an antibody against MMP12, an aptamer against MMP12, or a peptide having a site cleavable by MMP12. Item 18. A method for screening for a substance effective in treating an immune-mediated inflammatory disease characterized by increased MMP12 expression, the method comprising: measuring the MMP12 protein level in a biological sample obtained from a subject after administration of a test substance; and selecting the test substance as a candidate substance effective in treating an immune-mediated inflammatory disease characterized by increased MMP12 expression if the MMP12 protein level is reduced by administration of the test substance. Item 19. A therapeutic agent for an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising an MMP12 inhibitor.

[0008] According to the method, diagnostic agent, and diagnostic kit for detecting immune-mediated inflammatory diseases characterized by increased expression of MMP12 of the present invention, the disease can be detected or diagnosed using MMP12 with accuracy comparable to or higher than that of conventional prediction methods. The diagnostic agent and diagnostic kit do not use IL-6 as an indicator and can be used even under IL-6 inhibitory treatment.

[0009] Furthermore, the method of the present invention for screening for a substance effective in treating an immune-mediated inflammatory disease characterized by increased expression of MMP12 makes it possible to effectively screen for a therapeutic agent for the disease.

[0010] Furthermore, the therapeutic agent for immune-mediated inflammatory diseases of the present invention, which is characterized by increased expression of MMP12, can treat the diseases.

[0011] Graphs of ROC analysis. (A) Comparison between a group of patients with vasculitis syndrome and a group of healthy controls. (B) Comparison between a group of patients with vasculitis syndrome and a group of patients with immune-mediated inflammatory diseases other than vasculitis syndrome. Graphs showing serum MMP12 levels in each patient group. IBD: inflammatory bowel disease, TAK: Takayasu arteritis, NTM: nontuberculous mycobacterial infection, IgG4RD: IgG4 related disease, PAN: polyarteritis nodosa, MPA: microscopic polyangiitis, GPA: granulomatosis with polyangiitis, EGPA: eosinophilic granulomatosis with polyangiitis, GCA: giant cell arteritis. Expression of MMP12 in the aorta of patients with Takayasu arteritis. (A) HE staining, (B) enlarged photograph of the boxed area in Figure 3(A), (C) immunohistochemical staining for MMP12, (D) enlarged photograph of the boxed area in Figure 3(C). Expression of MMP12 in the temporal artery of a patient with giant cell arteritis. (A) HE staining, (B) enlarged photograph of the boxed area in Figure 4(A), (C) immunohistochemical staining for MMP12, (D) enlarged photograph of the boxed area in Figure 4(C). Expression of MMP12 in the lung of a patient with polyangiitis and granulomatosis. (A) HE staining, (B) enlarged photograph of the boxed area in Figure 5(A), (C) immunohistochemical staining for MMP12, (D) enlarged photograph of the boxed area in Figure 5(C). Graph showing the correlation between PETVAS and MMP12 at the time of diagnosis of Takayasu's arteritis and giant cell arteritis. Graphs showing the relationship between PETVAS values ​​and serum MMP12 levels and relapse rate. (A) Changes in MMP12 values ​​over time before and after treatment in a patient with monotonically declining type. (B) Image of the vascular wall before treatment in one of the male patients with monotonically declining type Takayasu's arteritis, (C) Image of the vascular wall of the same patient as in Figure 8(B) 8 months after the start of treatment. (A) Changes in MMP12 values ​​over time before and after treatment in a patient with monotonically declining type.(B) Image of the vascular wall of a male patient with smoldering Takayasu's arteritis before treatment. (C) Image of the vascular wall of the same patient as in Figure 9(B) 6 months after the start of treatment. (A) Changes in MMP12 levels over time before and after treatment in a patient with relapsing Takayasu's arteritis. (B) Image of the vascular wall of a male patient with Takayasu's arteritis before treatment. (C) Image of the vascular wall of the same patient as in Figure 10(B) at remission 1 month after the start of treatment. (D) Image of the vascular wall of the same patient as in Figure 10(B) at relapse 4 months after the start of treatment. Survival curves for mice in the non-MMP-administered group and the MMP-administered group. Urinary occult blood samples from mice in the MMP-administered group at 10, 15, and 20 weeks. Sequences and cleavage sites of n=22 positive controls and candidate peptides No. 1 to 10. Sequences and cleavage sites of candidate peptides No. 11 to 23.

[0012] As used herein, the singular forms "a," "an," and "the" are intended to include both the singular and the plural unless otherwise expressly stated herein or clearly contradicted by context.

[0013] In this specification, the terms "contain" and "comprise" are concepts that also encompass "consist only of."

[0014] In the numerical ranges described in stages in this specification, the upper or lower limit of a certain numerical range can be arbitrarily combined with the upper or lower limit of another numerical range. Furthermore, in the numerical ranges described in this specification, the upper or lower limit of the numerical range may be replaced with a value shown in an example or a value that can be unambiguously derived from an example. Furthermore, in this specification, a numerical value connected with "to" means a numerical range that includes the numbers before and after "to" as the upper and lower limits.

[0015] As used herein, "MMP12" refers to matrix metalloproteinase 12 protein. Human MMP12 is a 470-amino acid protein represented by NCBI Reference Sequence: NP#002417 (Uniprot P39900), and mouse MMP12 is a 473-amino acid protein represented by NCBI Reference Sequence: NP#032631 (Uniprot 34690). MMP12 is sometimes referred to as MMP-12, and these terms can be used interchangeably.

[0016] As used herein, "immune-mediated inflammatory diseases characterized by increased MMP12 expression" include vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), sarcoidosis, tuberculous mycobacterial disease, and nontuberculous mycobacterial disease. Vasculitis is a general term for diseases in which inflammation is observed in the blood vessels themselves and is also called vasculitis syndrome. Vasculitis syndromes are classified into three categories based on the size of the blood vessels: large-vasculitis, medium-sized vasculitis, and small-vasculitis. Large-vasculitis includes Takayasu arteritis (TAK) and giant cell arteritis (GCA). Medium-sized vasculitis includes polyarteritis nodosa and Kawasaki disease. Small-vessel vasculitis includes eosinophilic granulomatosis with polyangiitis (EGPA), granulomatosis with polyangiitis (GPA), and microscopic polyangiitis (MPA), which are classified as ANCA (anti-neutrophil cytoplasmic antibody)-associated vasculitis.

[0017] As used herein, "detection" of an immune-mediated inflammatory disease characterized by increased expression of MMP12 includes the diagnosis of the disease, the determination of patients (responders) who will respond to a therapeutic agent (companion diagnostic method), the determination of the preventive effect of the disease, the determination of the therapeutic effect of the disease, tests to assist in the diagnosis of the disease, and tests for the treatment of the disease (particularly early treatment).

[0018] As used herein, "assessing" an immune-mediated inflammatory disease characterized by increased expression of MMP12 includes not only determining whether or not the disease has developed, but also preventatively determining the possibility of the disease developing, predicting the prognosis of the disease after treatment, determining the possibility of remission, determining the possibility of relapse, assessing the timing of discontinuing drug administration, and determining the therapeutic effect of a therapeutic agent for the disease.

[0019] As used herein, the term "increased expression of MMP12" in "immune-mediated inflammatory disease characterized by increased expression of MMP12" refers to an increase in the level of MMP12 in a biological sample obtained from a subject compared to either the level of MMP12 in the subject before the onset of the immune-mediated inflammatory disease or the average level of MMP12 in healthy individuals.

[0020] As used herein, the term "immune-mediated inflammatory disease" refers to a chronic inflammatory disease in which the immune system causes damage to tissues through an immune response (e.g., a B cell response or a T cell response) to antigens that are part of the normal host (i.e., self-antigens) or without an immune response to antigens.

[0021] As used herein, the term "treatment" means curing or ameliorating a disease or symptom, or suppressing a symptom, and includes "prevention." "Prevention" means preventing the onset of a disease or symptom.

[0022] As used herein, the term "subject" refers to a mammal, including humans, mice, rats, cows, horses, pigs, monkeys, dogs, cats, rabbits, goats, sheep, etc., preferably humans and mice, and more preferably humans.

[0023] As used herein, the term "biological sample" may refer to a bodily fluid, tissue, or cell. Bodily fluids include blood (e.g., whole blood), blood cells, serum, plasma, pleural effusion, peritoneal fluid, cerebrospinal fluid, saliva, urine, stool, etc. Tissues include blood vessels from the heart, arteries, kidneys, lungs, inner ear, sinuses, skin, nerves, and other organs. Biological samples may be obtained directly from a patient or may be pretreated by other methods known in the art, such as filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the characteristics of the sample.

[0024] As used herein, the "level" of MMP12 can be the amount (eg, expression level) or concentration of MMP12 protein.

[0025] The inventors identified multiple molecules specific to vasculitis through cross-disease serum proteome analysis. Using the identified molecules, they compared a group of patients with vasculitis syndrome with a group of healthy controls, and a group of patients with vasculitis syndrome with a group of patients with immune-mediated inflammatory diseases other than vasculitis syndrome. They found that MMP12 had the highest sensitivity for ROC curve assessment. MMP12 levels were normal in patients with common bacterial infections and viral infections, and MMP12 could also be used to distinguish these from vasculitis syndrome.

[0026] Next, we investigated the expression of MMP12 in vasculitis tissues of Takayasu's arteritis (TAK), giant cell arteritis (GCA), and granulomatosis with polyangiitis (GPA), which are immune-mediated inflammatory diseases characterized by increased expression of MMP12. We confirmed its expression in the aorta of patients with Takayasu's arteritis, in the temporal artery of patients with giant cell arteritis, and in the lungs of patients with granulomatosis with polyangiitis, confirming its expression in vasculitis tissues.

[0027] Thus, the inventors have discovered that the expression of MMP12 is associated with the onset of immune-mediated inflammatory diseases characterized by increased expression of some MMP12s, and that MMP12 can be used as a diagnostic marker for immune-mediated inflammatory diseases characterized by increased expression of MMP12. They have also discovered a diagnostic agent for immune-mediated inflammatory diseases characterized by increased expression of MMP12, a method for screening substances effective in treating immune-mediated inflammatory diseases characterized by increased expression of MMP12, and a therapeutic agent for immune-mediated inflammatory diseases characterized by increased expression of MMP12.

[0028] According to a first aspect of the present invention, there is provided a method for detecting an immune-mediated inflammatory disease in a subject characterized by increased expression of MMP12, the method comprising measuring the level of MMP12 in a biological sample obtained from the subject.

[0029] According to this method, an examiner can easily and accurately detect immune-mediated inflammatory diseases in vitro based on the test results. Furthermore, this method does not include diagnostic methods for humans, which are medical procedures.

[0030] The method for measuring the level of MMP12 in a biological sample obtained from a subject is not particularly limited, and measurement can be performed using a substance that specifically interacts with MMP12. Examples of substances that specifically interact with MMP12 include substances that bind to MMP12 and substances that are cleaved by MMP12 due to the protease activity of MMP12.

[0031] In some embodiments, the level of MMP12 in a biological sample obtained from a subject may be measured by known protein identification methods, including, but not limited to, immunoassays and mass spectrometry. Immunoassays include, but are not limited to, enzyme-linked immunosorbent assays (e.g., ELISA), fluorescent antibody assays, radioimmunoassays, solid-phase assays, and sandwich assays. For example, a substance that binds to MMP12 is bound to the MMP to form a complex, and the signal obtained depending on the amount of the complex is detected directly or indirectly.

[0032] In some embodiments, the method further comprises contacting a biological sample obtained from the subject with a substance that binds to MMP12, and measuring the level of MMP12 comprises measuring the level of a complex between MMP12 and the substance that binds to MMP12. Preferably, the substance that binds to MMP12 is a substance that specifically binds to MMP12. Note that "specifically bind" refers to a bond that is measurably different from a non-specific interaction, and refers to binding to each other.

[0033] In some embodiments, the substance that binds to MMP12 can be selected from the group consisting of an antibody against MMP12, an antigen-binding fragment of an antibody against MMP12, and an aptamer against MMP12.

[0034] MMP12 and / or substances that bind to MMP12 may be further conjugated to radioisotopes, enzymes, fluorescent dyes, and contrast agents (eg, paramagnetic ions) as detection labels or imaging probes.

[0035] An antibody against MMP12, i.e., an anti-MMP12 antibody, can be produced using a known method for producing an antibody.

[0036] Methods for producing antibodies are well known (see, for example, Antibodies: A Laboratory Manual (1988) by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Examples of antibodies include polyclonal antibodies, monoclonal antibodies, and modified antibodies such as chimeric antibodies and humanized antibodies.

[0037] For example, antibody production can be induced by immunizing a host animal with the full-length MMP12 polypeptide or a fragment thereof as an immunogen, i.e., epitope. When an MMP12 fragment is used, the region of the polypeptide that serves as the immunogen can be selected arbitrarily, or, since many examples of the preparation of anti-MMP12 antibodies, including commercially available products, have been published, these published examples can be used as a reference. The immunogenic polypeptide can be produced by known standard methods, and can be chemically synthesized or biochemically synthesized as a recombinant protein. It can also be obtained by using commercially available custom synthesis services.

[0038] To produce anti-MMP12 polyclonal antibodies, the immunogen polypeptide itself may be used for immunization, or a reconstituted membrane or recombinant cells displaying a recombinant protein containing the polypeptide may be used for immunization. The host animal to be immunized is not particularly limited, but animal species such as mouse, rat, rabbit, guinea pig, sheep, goat, donkey, chicken, or camel are preferred. Mouse or rat is more preferred, and mouse is particularly preferred. Antisera containing anti-MMP12 antibodies can be prepared by known standard methods. The antibody may be of any of the five classes of immunoglobulin molecules (IgG, IgM, IgA, IgD, and IgE). IgG or IgM is preferred, and IgG is more preferred.

[0039] Anti-MMP12 monoclonal antibodies can be prepared by fusing the antibody-producing cells obtained in the above preparation process with myeloma cells and then cloning the monoclonal antibody. Alternatively, they can be produced by expressing chemically synthesized antibody genes in Escherichia coli or other organisms using genetic engineering techniques. The methods for fusing antibody-producing cells with myeloma cells, screening for desired cells from a cell population containing fused cells, monocloning the cells selected by screening, and preparing monoclonal antibodies from clones can all be performed using known standard methods. The desired monoclonal antibody can also be synthesized based on sequence information using known standard methods.

[0040] The MMP12 monoclonal antibody can be artificially modified to produce a recombinant antibody, for example, to reduce heterologous antigenicity to humans. Examples of such antibodies include chimeric antibodies and humanized antibodies. These modified antibodies can be produced by known methods.

[0041] A chimeric antibody can be prepared by linking DNA encoding the variable (V) region of the anti-MMP12 monoclonal antibody of this embodiment to DNA encoding the constant (C) region of a human antibody, incorporating this into an expression vector, and introducing it into a host for production.

[0042] A humanized antibody is one in which the CDRs of a non-human mammal, such as a mouse antibody, are grafted onto the CDRs of a human antibody (CDR grafting). Humanized antibodies can be prepared using standard genetic recombination techniques. For example, a DNA sequence designed to encode an amino acid sequence linking each CDR of a mouse anti-MMP12 monoclonal antibody to the framework region of a human antibody can be synthesized by PCR using multiple oligonucleotides as primers that have overlapping regions at the terminal regions of both the CDRs and FRs.

[0043] The FRs of the variable regions of human antibodies can be obtained from published DNA databases, etc. The constant regions of chimeric antibodies and humanized antibodies can be those of human antibodies. For example, Cγ1, Cγ2, Cγ3, and Cγ4 may be used for the heavy chain, and Cκ and Cλ may be used for the light chain.

[0044] An antigen-binding fragment of an antibody refers to a fragment containing the antigen-binding portion of an antibody molecule. As long as it exhibits sufficient specificity and affinity to confer specific antigen binding, a substance that binds to MMP12 does not necessarily have to maintain the structure of the entire immunoglobulin molecule. Because the antigen-binding ability of an antibody is governed by the variable region of the antibody, the constant region of the immunoglobulin molecule does not necessarily need to be present. Therefore, antigen-binding fragments of antibodies of this embodiment include fragments consisting of the variable region of an immunoglobulin molecule, such as Fab, Fab', F(ab')2, Fd (Fab with the VL removed), single-chain Fv fragments (scFv), and dimers thereof, such as bispecific antibodies (diabodies).

[0045] Aptamers are oligonucleotides that bind to their targets with high affinity and specificity. Aptamers are a class of molecules that replace antibodies in molecular recognition, and aptamers for MMP12 are oligonucleotides that have the ability to recognize MMP12 with high affinity and specificity. As is well known to those skilled in the art, aptamers can be obtained as substances that specifically bind to target compounds by known methods that involve repeated in vitro processes including "selection and amplification," such as the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (see, for example, JP 2006-320289 A, JP 2009-207491 A, or JP 2009-165394 A).

[0046] Nucleic acid aptamers as substances that bind to MMP12 can be selected by using MMP12 as a target compound, typically the full-length polypeptide or a fragment thereof of human MMP12. The nucleic acid aptamer used in this embodiment may have any binding activity as long as it specifically binds to the target compound MMP12 and functions as an MMP12 inhibitor through said binding, but it has a binding ability that shows a dissociation constant (KD) of 2 μM or less, preferably 600 nM or less, for human MMP12.

[0047] Here, the dissociation constant (KD) in this specification refers to a constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed in molar concentration (M). The KD value of a nucleic acid aptamer can be determined using a method established in the art. A preferred method for determining the KD of a nucleic acid aptamer is to use surface plasmon resonance, preferably using a biosensor system such as a Biacore® system.

[0048] In some embodiments, the method further comprises contacting a biological sample obtained from the subject with a substance that is cleaved by MMP12, and measuring the level of MMP12 comprises measuring a change (e.g., fluorescence) caused by cleavage of the substance that is cleaved by MMP12. The substance that binds to MMP12 includes, for example, a peptide having a site that can be cleaved by MMP12. The diagnostic agent of the third aspect described below can be used as the substance that is cleaved by MMP12. In some embodiments, the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitic syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis.

[0049] In some embodiments, the immune-mediated inflammatory disease characterized by increased expression of MMP12 is granulomatous vasculitis.

[0050] In some embodiments, the biological sample is blood, serum, or plasma.

[0051] The method for detecting an immune-mediated inflammatory disease characterized by increased expression of MMP12 according to the first aspect can be used in various tests or assessments.

[0052] In some embodiments, the method is a method for predicting the likelihood of developing an immune-mediated inflammatory disease characterized by increased expression of MMP12 in a subject, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value, and if the level of MMP12 in the biological sample obtained from the subject is higher than the reference value, it indicates that the subject is likely to develop an immune-mediated inflammatory disease characterized by increased expression of MMP12.

[0053] Examples of the reference value include an average or median value calculated from measurements of MMP12 levels in multiple healthy individuals, a cutoff value (e.g., within a range of 50 pg / ml to 200 pg / ml, preferably 100 pg / ml) that separates a group of healthy individuals from a group of patients diagnosed by a doctor as suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression, or a certain value (e.g., within a range of 250 pg / ml to 1000 pg / ml, preferably 500 pg / ml) that is higher than the average or median value calculated from measurements of MMP12 levels in multiple healthy individuals and lower than the average or median value calculated from measurements of MMP12 levels in multiple patients suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression.

[0054] If it is determined by such criteria that a subject is highly likely to develop an immune-mediated inflammatory disease characterized by increased expression of MMP12, this determination result can be taken into consideration when determining the subject's future treatment policy, such as performing additional diagnoses of immune-mediated inflammatory diseases. The method of this embodiment is useful as a means for diagnosing (particularly early diagnosis) or assisting in diagnosis of the possibility of developing an immune-mediated inflammatory disease.

[0055] In some embodiments, the method is a method for predicting the likelihood of a subject developing an immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value, and if the level of MMP12 in the biological sample obtained from the subject is equal to or lower than the reference value, this indicates that the subject is unlikely to develop an immune-mediated inflammatory disease characterized by increased expression of MMP12.

[0056] The reference value may be the average or median calculated from measurements of MMP12 levels in multiple healthy individuals, or a value lower than these.

[0057] If such criteria determine that a subject is unlikely to develop an immune-mediated inflammatory disease characterized by increased expression of MMP12, this determination result can be taken into consideration when determining the subject's future treatment plan.

[0058] In some embodiments, the method is a method for predicting the prognosis of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the subject has been treated for the immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value, and if the level of MMP12 in the biological sample obtained from the subject is higher than the reference value, this indicates a high possibility of relapse of the immune-mediated inflammatory disease characterized by increased expression of MMP12 in the subject.

[0059] Subjects who have been treated for an immune-mediated inflammatory disease characterized by increased expression of MMP12 also include patients who have been treated for an immune-mediated inflammatory disease characterized by increased expression of MMP12 and then discontinued the treatment.

[0060] Treatments include administration of drugs useful for treating immune-mediated inflammatory diseases characterized by increased expression of MMP12, radiation therapy, exercise therapy, dietary therapy, administration of supplements, and the like.

[0061] Examples of the reference value include the level of MMP12 in a sample obtained from the same subject before undergoing treatment for treating an immune-mediated inflammatory disease characterized by increased MMP12 expression; a cutoff value (e.g., within the range of 50 pg / ml to 200 pg / ml, preferably 100 pg / ml) that distinguishes between a group of healthy subjects and a group of patients diagnosed by a doctor as suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression; or a certain value (e.g., within the range of 250 pg / ml to 1000 pg / ml, preferably 500 pg / ml) that is higher than the mean or median calculated from measurements of the MMP12 levels of multiple healthy subjects and lower than the mean or median calculated from measurements of the MMP12 levels of multiple patients suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression.

[0062] If such criteria are used to determine that a subject is likely to have a relapse of an immune-mediated inflammatory disease characterized by increased MMP12 expression, this determination result can be taken into consideration when determining the subject's future treatment policy, such as resuming administration of a therapeutic drug. Treatment for the subject can be more appropriately administered. Because an increase in MMP12 levels precedes the appearance of symptoms related to the relapse of an immune-mediated inflammatory disease, the method of this embodiment is useful as a means for diagnosing (particularly early diagnosis) or assisting in diagnosis of the possibility of a relapse of an immune-mediated inflammatory disease.

[0063] The present inventors have demonstrated that MMP12 is positively correlated with the PET vascular activity score (PETVAS) used in PET-CT examinations. The method of this embodiment may be a superior evaluation tool to PETVAS and is useful for stratifying the risk of relapse in subjects.

[0064] In some embodiments, the method is a method for predicting the prognosis of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the subject has been treated for the immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value, and if the level of MMP12 in the biological sample obtained from the subject is equal to or lower than the reference value, this indicates a low likelihood of relapse of the immune-mediated inflammatory disease characterized by increased expression of MMP12 in the subject.

[0065] Subjects who have been treated for an immune-mediated inflammatory disease characterized by increased expression of MMP12 also include patients who have been treated for an immune-mediated inflammatory disease characterized by increased expression of MMP12 and then discontinued the treatment.

[0066] Treatments include administration of drugs useful for treating immune-mediated inflammatory diseases characterized by increased expression of MMP12, radiation therapy, exercise therapy, dietary therapy, administration of supplements, and the like.

[0067] Examples of the reference value include the level of MMP12 in a sample obtained from the same subject before undergoing treatment for treating an immune-mediated inflammatory disease characterized by increased MMP12 expression; a cutoff value (e.g., in the range of 50 pg / ml to 200 pg / ml, preferably 100 pg / ml) that distinguishes between a group of healthy subjects and a group of patients diagnosed by a physician as suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression; or a certain value (e.g., in the range of 250 pg / ml to 1000 pg / ml, preferably 500 pg / ml) that is higher than the mean or median calculated from measurements of MMP12 levels in multiple healthy subjects and lower than the mean or median calculated from measurements of MMP12 levels in multiple patients suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression. Hereinafter, the "cutoff value" can be determined by various statistical analysis methods well known to those skilled in the art. Examples of such a value include the average or median level of MMP12 in patients suffering from an immune-mediated inflammatory disease characterized by an increase in the expression of MMP12 in several individuals; the value at which the effect of the above-mentioned treatment to separate the patient group from the healthy subject group is at its smallest P value in a log-rank test or at which the P value is below a certain level (for example, a value at which the P value is less than 0.1, a value at which the P value is less than 0.05); a value determined based on ROC (Receive Operating Characteristic) analysis so as to maximize the sum of sensitivity and specificity from the relationship between the expression level of MMP12 in the treated patient and the effect of the treatment; and the value at which the effect of the treatment to separate the healthy subject group from the patient group is at its smallest P value in a chi-square test or at which the P value is below a certain level (for example, a value at which the P value is less than 0.1, a value at which the P value is less than 0.05).

[0068] If such criteria are used to determine that a subject is unlikely to experience a relapse of an immune-mediated inflammatory disease characterized by increased expression of MMP12, this determination result can be taken into consideration when deciding on the subject's future treatment plan, such as refraining from further treatment, which is advantageous from the perspective of reducing the burden on the subject and from the economic perspective.

[0069] In some embodiments, the method is a method for predicting the post-treatment status of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the subject has been treated for the immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value, and if the level of MMP12 in the biological sample obtained from the subject is lower than the reference value, this indicates that the immune-mediated inflammatory disease characterized by increased expression of MMP12 in the subject is likely to be in remission.

[0070] Treatments include administration of drugs useful for treating immune-mediated inflammatory diseases characterized by increased expression of MMP12, radiation therapy, exercise therapy, dietary therapy, administration of supplements, and the like.

[0071] Examples of drugs useful for treating immune-mediated inflammatory diseases characterized by increased expression of MMP12 include, but are not limited to, steroids (Prednisolone®), cyclophosphamide (Endoxan®), methotrexate (Rheumatrex®), rituximab (Rituxan®), avacopan (Taboones®), and tocilizumab (Actemra®).

[0072] Examples of the reference value include the level of MMP12 in a sample obtained from the same subject before undergoing treatment for treating an immune-mediated inflammatory disease characterized by increased MMP12 expression; a cutoff value (e.g., within the range of 50 pg / ml to 200 pg / ml, preferably 100 pg / ml) that distinguishes between a group of healthy subjects and a group of patients diagnosed by a doctor as suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression; or a certain value (e.g., within the range of 250 pg / ml to 1000 pg / ml, preferably 500 pg / ml) that is higher than the mean or median calculated from measurements of the MMP12 levels of multiple healthy subjects and lower than the mean or median calculated from measurements of the MMP12 levels of multiple patients suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression.

[0073] If such criteria are used to determine that a subject is likely to be in remission from an immune-mediated inflammatory disease characterized by increased expression of MMP12, the effectiveness of the treatment received by the subject can be evaluated, and this determination result can be taken into consideration in determining the subject's future treatment plan, such as withholding the administration of therapeutic drugs, which is advantageous from the perspective of the burden on the subject and economics.

[0074] In some embodiments, the method is for evaluating the timing of administration of a therapeutic agent for an immune-mediated inflammatory disease characterized by increased expression of MMP12 to a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value, and a lower level of MMP12 in the biological sample obtained from the subject compared to the reference value indicates the need to discontinue administration of the drug to the subject.

[0075] The above-mentioned reference value may be a cutoff value (e.g., within a range of 50 pg / ml to 200 pg / ml, preferably 100 pg / ml) that distinguishes between a group of healthy subjects and a group of patients diagnosed by a doctor as suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression, or a certain value (e.g., within a range of 250 pg / ml to 1000 pg / ml, preferably 500 pg / ml) that is higher than the mean or median value calculated from measurements of MMP12 levels in multiple healthy subjects and lower than the mean or median value calculated from measurements of MMP12 levels in multiple patients suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression.

[0076] Using such criteria, if the level of MMP12 in a biological sample obtained from a subject is lower than the reference value, it is determined that it is appropriate to suspend administration of the drug to the subject, and this determination result can be taken into consideration when deciding on the subject's future treatment plan, such as refraining from administering therapeutic drugs, which is advantageous from the perspective of burden on the subject and economics.

[0077] In some embodiments, the method is a method for determining the therapeutic effect of a treatment in a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the subject has been treated for an immune-mediated inflammatory disease characterized by increased expression of MMP12, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value; if the level of MMP12 in the biological sample obtained from the subject is lower compared to the reference value, this indicates that the therapeutic effect of the immune-mediated inflammatory disease characterized by increased expression of MMP12 due to the treatment in the subject is high.

[0078] Treatments include administration of drugs useful for treating immune-mediated inflammatory diseases characterized by increased expression of MMP12, radiation therapy, exercise therapy, dietary therapy, administration of supplements, and the like.

[0079] Examples of the reference value include the level of MMP12 in a sample obtained from the same subject before undergoing treatment for treating an immune-mediated inflammatory disease characterized by increased MMP12 expression; a cutoff value (e.g., within the range of 50 pg / ml to 200 pg / ml, preferably 100 pg / ml) that distinguishes between a group of healthy subjects and a group of patients diagnosed by a physician as suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression; or a certain value (e.g., within the range of 250 pg / ml to 1000 pg / ml, preferably 500 pg / ml) that is higher than the mean or median calculated from measurements of the MMP12 levels of multiple healthy subjects and lower than the mean or median calculated from measurements of the MMP12 levels of multiple patients suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression.

[0080] If such criteria are used to determine that the treatment of an immune-mediated inflammatory disease characterized by increased MMP12 expression in a subject is highly effective, the effectiveness of the treatment received by the subject can be evaluated, and this determination result can be taken into consideration in determining the subject's future treatment plan, such as withholding the administration of therapeutic drugs, which is advantageous from the perspective of reducing the burden on the subject and from the economic standpoint.

[0081] According to a second aspect of the present invention, there is provided a biomarker for diagnosing or detecting immune-mediated inflammatory diseases characterized by increased expression of MMP12, including MMP12.

[0082] In some embodiments, MMP12 in a biological sample obtained from a subject is used as a biomarker for the diagnosis or detection of immune-mediated inflammatory diseases characterized by increased expression of MMP12.

[0083] According to a third aspect of the present invention, there is provided a diagnostic agent for immune-mediated inflammatory diseases characterized by increased expression of MMP12, which comprises a substance that specifically interacts with MMP12. The term "specifically interact" refers to an interaction that is measurably different from a non-specific interaction, and refers to an interaction between two substances.

[0084] Substances that specifically interact with MMP12 include substances that bind to MMP12, as described in the first embodiment, and substances that are cleaved by MMP12 due to the protease activity of MMP12, such as peptides having a site that can be cleaved by MMP12.

[0085] In some embodiments, the diagnostic agent comprises a substance that binds to MMP12, and the substance that binds to MMP12 can be selected from the group consisting of an antibody against MMP12, an antigen-binding fragment of an antibody against MMP12, and an aptamer against MMP12.

[0086] In some embodiments, the diagnostic agent utilizes the protease activity of MMP12 and includes a conjugate of a peptide having a site cleavable by MMP12 as a substrate site for MMP12 and a labeling substance. The peptide having a site cleavable by MMP12 and the labeling substance may be directly linked or linked via a linker. The peptide having a site cleavable by MMP12 and the labeling substance can be linked by any known method, with or without a linker. Linkers can also be interchangeably referred to as "spacers." Examples of linkers include, but are not limited to, saturated hydrocarbon groups having 5 to 12 carbon atoms or 1 to 5 amino acids, which may have a substituent. The linker can be linked to the N-terminus, C-terminus, or both of the peptide. Linkers that separate the peptide recognized by the protein to be detected and the labeling substance are well known, and those skilled in the art can easily select such linkers. The diagnostic agent may be an in vitro diagnostic agent or an in vivo diagnostic agent.

[0087] The length of the peptide having a site cleavable by MMP12 is not particularly limited, but from the viewpoint of MMP12 detection sensitivity, it is preferably composed of an amino acid sequence of 8 to 25 amino acids, more preferably 10 to 20 amino acids, and even more preferably 12 to 18 amino acids. The peptide having a site cleavable by MMP12 can be determined based on the peptide sequence of a protein known to be a selective substrate of MMP12. Proteins known to be selective substrates of MMP12 include, for example, human CCL-14 (UniPlot Q16627), mouse CXCL1 (UniPlot A2RTH0), mouse CXCL2 (UniPlot P12850), mouse CXCL3 (UniPlot P10889), mouse CXCL5 (UniPlot P50228), human CXCL1 (UniPlot P09341), human CXCL2 (UniPlot P19875), human CXCL3 (UniPlot P19876), human CXCL5 (UniPlot P42830), human CXCL6 (P80162), human CXCL7 (UniPlot P02775), human CXCL8 (UniPlot P10145), human INF-α-2 (UniPlot P01563), and human INF-α-6 (UniPlot P10145). A0A0A0MQU8), human IFN-α-8 (UniPlot P32881), human IFN-α-10 (UniPlot P01566), human IFN-γ (UniPlot P01579), mouse IFN-γ (UniPlot P01580), etc. See, for example, J Biol Chem 2012, 287, 5848-60, Blood 2008, 112, 3455-64, Nat Med. 2014, 20, 493-502, Nat Commun. 2018, 9, 2416.In some embodiments, the peptide having a site cleavable by MMP12 is selected from the group consisting of human CCL-14 (hCCL-14), mouse CXCL1 (mCXCL1), mouse CXCL2 (mCXCL2), mouse CXCL3 (mCXCL3), mouse CXCL5 (mCXCL5), human CXCL1 (hCXCL1), human CXCL2 (hCXCL2), human CXCL3 (hCXCL3), human CXCL5 (hCXCL5), human CXCL6 (hCXCL6), human CXCL7 (hCXCL7), human CXCL8 (hCXCL8), human INF-α-2 (hINF-α-2), human INF-α-6 (hINF-α-6), and human INF-α- The peptide comprises a sequence of 8 to 25 consecutive amino acids, preferably 10 to 20 amino acids, and more preferably 12 to 18 amino acids, containing an amino acid sequence having a site cleavable by MMP12 in a protein selected from human IFN-α-8 (hIFN-α-8), human IFN-α-10 (hIFN-α-10), human IFN-γ (hIFN-γ), and mouse IFN-γ (mIFN-γ). However, an amino acid that is cysteine ​​(Cys) in the native sequence of the peptide may be substituted with another amino acid such as serine (Ser).

[0088] Sites in peptides that are specifically recognized and cleavable by MMP12 are known, and examples thereof include, but are not limited to, ELRCXC (cleavage between E and LRCXC, where X is preferably Q or V), PLGLAG (cleavage between PLG and LAG, see J. Am. Chem. Soc., 2012, 134, 13730-1373), PLGLR (cleavage between LG and LR, see Chemistry and Biology, 2015, 22, 1122-1133), FGALT (cleavage between FGA and LT, see above), VYDLK (cleavage between VYD and LK, see above), and PWAWR (cleavage between PWA and WR, see above).

[0089] From the viewpoint of specificity for MMP12, it is preferable that the amino acid sequence of the peptide having a site cleavable by MMP12 is the same as or similar to a natural amino acid sequence.

[0090] Examples of peptides having a site cleavable by MMP12 that is the same as or similar to a natural amino acid sequence include partial peptides containing the ELRCXC sequence of CXC (α-chemokine), a subfamily of chemokine proteins, or variants thereof.

[0091] In some embodiments, the peptide having a site cleavable by MMP12 is a peptide consisting of an 8 to 25 amino acid sequence having the amino acid sequence ELRCXC (X is Q or V). Sequences upstream and downstream of ELRCXC can be designed by a person skilled in the art using ordinary skill based on the amino acid sequence of a CXC protein, provided that the peptide has the ability to be cleaved by MMP12.

[0092] In some embodiments, the peptide having a site cleavable by MMP12 is either (i) or (ii) below: (i) human CXCL1 (UniPlot P09341), human CXCL2 (UniPlot P19875), human CXCL3 (UniPlot P19876), human CXCL5 (UniPlot P42830), human CXCL8 (UniPlot P10145), mouse CXCL1 (UniPlot A2RTH0), mouse CXCL2 (UniPlot P12850), mouse CXCL3 (UniPlot P10889), mouse CXCL5 (UniPlot (ii) a peptide having a total length of 8 to 25 amino acids, which is a partial sequence of any of the amino acid sequences of ELRCXC (X is Q or V), at least 1 to 5 amino acids upstream thereof, and one or more amino acids downstream thereof, and which is the amino acid sequence of ELRCXC (X is Q or V), (iii) a peptide having a total length of 8 to 25 amino acids, which is the peptide of (i) above, in which one or more amino acids upstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted, or one or more amino acids downstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted, or one or more amino acids upstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted and one or more amino acids downstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted.

[0093] In some embodiments, the peptide having a site cleavable by MMP12 is any of the following (i) to (vi): (i) a peptide having the amino acid sequence of any of SEQ ID NOs: 1 to 9; (ii) a peptide consisting only of the amino acid sequence of any of SEQ ID NOs: 1 to 9; (iii) a peptide consisting of a partial sequence of a peptide having the amino acid sequence of any of SEQ ID NOs: 1 to 9, which has the portion ELRCXC (X is Q or V), or (iv) a peptide of the above (i) to (iii), in which one or more of the amino acids at positions 1 to 5 of the amino acid sequence of any of SEQ ID NOs: 1 to 9 have been substituted, one or more of the amino acids at positions 12 to 13 of the amino acid sequence of any of SEQ ID NOs: 1 to 9 have been substituted, or both one or more of the amino acids at positions 1 to 5 of the amino acid sequence of any of SEQ ID NOs: 1 to 9 and one or more of the amino acids at positions 12 to 13 of the amino acid sequence of any of SEQ ID NOs: 1 to 9 have been substituted.

[0094] Amino acid sequence of peptide derived from human CXCL1: ASVATELRCQCLQ (SEQ ID NO: 1) Amino acid sequence of peptide derived from human CXCL2: APLATELRCQCLQ (SEQ ID NO: 2) Amino acid sequence of peptide derived from human CXCL3: ASVVTELRCQCLQ (SEQ ID NO: 3) Amino acid sequence of peptide derived from human CXCL5: AAVLRELRCVCLQ (SEQ ID NO: 4) Amino acid sequence of peptide derived from human CXCL8: PRSAKELRCQCIK (SEQ ID NO: 5) Amino acid sequence of peptide derived from mouse CXCL1: APIANELRCQCLQ (SEQ ID NO: 6) Amino acid sequence of peptide derived from mouse CXCL2: AVVASELRCQCLK (SEQ ID NO: 7) Amino acid sequence of peptide derived from mouse CXCL3: AVVASELRCQCLN (SEQ ID NO: 8) Amino acid sequence of peptide derived from mouse CXCL5: VIAATELRCVCLT (SEQ ID NO: 9) In some embodiments, the peptide having a site cleavable by MMP12 is any one of the following (i) to (v): (i) a peptide having an amino acid sequence of any one of SEQ ID NOs: 10 to 32, (ii) a peptide consisting only of an amino acid sequence of any one of SEQ ID NOs: 10 to 32, (iii) a peptide consisting of a partial sequence of a peptide having an amino acid sequence of any one of SEQ ID NOs: 10 to 32 having a site cleavable by MMP12, the total length of which is 8 to 25 amino acids, (iv) a peptide in which one or two amino acids other than the amino acid having the site cleavable by MMP12 are substituted in any one of the peptides (i) to (iii) above, or (v) a peptide in which all cysteines in the native sequence are substituted with other amino acids such as serine in any one of the peptides (i) to (iii) above. Any one of the peptides (i) to (v) is a peptide consisting of a sequence of 8 to 25 amino acids, preferably a peptide consisting of a sequence of 10 to 20 amino acids, and more preferably a peptide consisting of a sequence of 12 to 18 amino acids. Amino acid sequence at positions 1-12 of hCCL-14: TKTESSSRGPYH (SEQ ID NO: 10) Possible cleavage sites are TE at positions 3-4 and SS at positions 6-7 Amino acid sequence at positions 61-74 of hCCL-14: DKWVQDYIKDMKEN(SEQ ID NO: 11) MK at positions 11-12 and KE at positions 12-13 are potential cleavage sites Amino acid sequence at positions 154-165 of hIFN-α-10 STNLQKRLRRKD (SEQ ID NO: 12) LQ at positions 4-5 and LR at positions 8-9 are potential cleavage sites Amino acid sequence at positions 150-161 of hIFN-α-10 SLSFSTNLQKRL (SEQ ID NO: 13) LQ at positions 8-9 is potential cleavage site Amino acid sequence at positions 154-165 of hIFN-α-6 SRNLQERLRRKE (SEQ ID NO: 14) LQ at positions 4-5 and LR at positions 8-9 are potential cleavage sites Amino acid sequence at positions 150-161 of hIFN-α-6 SFSSSRNLQERL (SEQ ID NO: 15) LQ at positions 8-9 is potential cleavage site Amino acid sequence of positions 154-165 of hIFN-α-2 STNLQESLRRKE (SEQ ID NO: 16) LQ at positions 4-5 and LR at positions 8-9 are potential cleavage sites Amino acid sequence of positions 150-161 of hIFN-α-2 SFSLSTNLQESL (SEQ ID NO: 17) LQ at positions 8-9 is potential cleavage site Amino acid sequence of positions 154-165 of hIFN-α-8 SINLQKRLKSKE (SEQ ID NO: 18) LQ at positions 4-5 and LK at positions 8-9 are potential cleavage sites Amino acid sequence of positions 150-161 of hIFN-α-8 SFSLSINLQKRL (SEQ ID NO: 19) LQ at positions 8-9 is potential cleavage site Amino acid sequence of positions 126-139 of hIFN-γ LNVQRKAIHELIQV (SEQ ID NO: 20) The cleavage site is EL at positions 10-11. Amino acid sequence of positions 130-143 of hIFN-γ: RKAIHELIQVMAEL (SEQ ID NO: 21). The cleavage site is EL at positions 6-7. Amino acid sequence of positions 151-165 of hIFN-γ: KRKRSQMLFRGRRAS (SEQ ID NO: 22). The cleavage site is ML at positions 7-8. Amino acid sequence of positions 125-137 of mIFN-γ: QVQRQAFNEKIRV (SEQ ID NO: 23). The cleavage site is EL at positions 9-10. Amino acid sequence of positions 128-141 of mIFN-γ: RQAFNEKIRVVHQL (SEQ ID NO: 24). The cleavage site is EL at positions 6-7. Amino acid sequence of positions 143-154 of mIFN-γ: PESSLRKRKRSR (SEQ ID NO: 25). RK at positions 8-9.Amino acid sequence of positions 1-14 of mCXCL1 APIANELRSQSLQT (SEQ ID NO: 26) Possible cleavage sites are PI at positions 2-3 and EL at positions 6-7 Amino acid sequence of positions 78-90 of mCXCL5 AKRNALAVERTAS (SEQ ID NO: 27) Possible cleavage site is AV at positions 7-8 Amino acid sequence of positions 76-89 of hCXCL7 APRIKKIVQKKLAG (SEQ ID NO: 28) Possible cleavage site is KI at positions 6-7 Amino acid sequence of positions 1-14 of hCXCL3 ASVVTELRSQSLQT (SEQ ID NO: 29) Possible cleavage site is EL at positions 6-7 Amino acid sequence of positions 3-14 of hCXCL8 LPRSAKELRSQS (SEQ ID NO: 30) Possible cleavage site is LR at positions 8-9 Amino acid sequence of positions 3-15 of hCXCL5 PAAAVLRELRSVS (SEQ ID NO: 31) EL at positions 8-9 is a cleavable site. The amino acid sequence of positions 9-22 of hCXCL6 is ELRSTSLRVTLRVN (SEQ ID NO: 32). SL at positions 6-7 is a cleavable site. In a particularly preferred embodiment, the peptide having a site cleavable by MMP12 is any one of the following (i) to (v): (i) a peptide having an amino acid sequence of any one of SEQ ID NOs: 11, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31; (ii) a peptide consisting only of an amino acid sequence of any one of SEQ ID NOs: 111, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31; (iii) a peptide consisting of a partial sequence of a peptide having any one of the amino acid sequences of SEQ ID NOs: 111, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31, which has a site cleaved by MMP12, and which has a total length of 8 to 25 amino acids; (iv) a peptide in which one or two amino acids other than the amino acid having the site cleaved by MMP12 are substituted in any one of the peptides (i) to (iii) above; and (v) a peptide in which all cysteines in the native sequence are substituted with other amino acids such as serine in any one of the peptides (i) to (iii) above.

[0095] Labeling substances include radioisotopes, enzymes, fluorescent dyes, and contrast agents (eg, paramagnetic ions).

[0096] Examples of radioisotopes include: 3 H], [ 11 C], [ 14 C], [ 18 F], [ 34 Cl], [ 38 Cl], [ 75 Br], [ 76 Br], [ 77 Br], [ 80 Br], [ 82 Br], [ 121 I], [ 123 I], [ 124 I], [ 125 I], [ 127 I], [ 131 I], [ 64 Cu], [ 90 Y], [ 67 Ga], [ 51 Cr], [ 192 Ir], [ 99 Mo], [ 153 Sm], [ 201 Tl] and the like.

[0097] Examples of fluorescent dyes include phenyl and its derivatives, naphthalene and its derivatives such as 5-dimethylaminonaphthalene-1-sulfonic acid and hydroxynaphthalenes, anthracene and its derivatives such as 9,10-diphenylnaphthalene and 9-methylanthracene, pyrene and its derivatives such as N-(1-pyrene)iodoacetamide and hydroxypyrenes, biphenyl and its derivatives, acridine and its derivatives such as hydroxyacridines and 9-methylacridine, coumarin and its derivatives such as 7-dialkylamino-4-methylcoumarin and 4-bromomethyl-7-methoxycoumarin, xanthene and its derivatives, phthalocyanine and its derivatives, stilbene and its derivatives such as 6,6'-dibromostilbene and hydroxystilbenes, furan and its derivatives, oxazole and its derivatives, oxadiazole and its derivatives, nitrobenzoxadiazole and its derivatives such as hydroxynitrobenzoxadiazoles, benzothiazole and its derivatives, fluorescein and 5-iodoacetamide These include, but are not limited to, midofluorescein and derivatives thereof such as fluorescein-5-maleimide, rhodamine and derivatives thereof such as tetramethylrhodamine, tetraethylrhodamine, carboxytetramethylrhodamine, BODIPY® and derivatives thereof, eosin and derivatives thereof, erythrosine and derivatives thereof such as hydroxyerythrosines and 5-iodoacetamidoerythrosine, resorufin and derivatives thereof such as hydroxyresorufin, quinoline and derivatives thereof such as 6-hydroxyquinoline and 6-aminoquinoline, carbazole and derivatives thereof such as N-methylcarbazole, cyanine and derivatives thereof such as hydroxycyanine, carbocyanine and derivatives thereof such as phenylcarbocyanine, pyridinium salts and derivatives thereof such as 4-(4-dialkyldiamidostyryl)-N-methylpyridinium iodate, fluorescent complexes of lanthanides and derivatives thereof, green fluorescent protein (GFP) and derivatives thereof, benzoindole and derivatives thereof (such as indocyanine green), etc. Derivatives are preferably compounds that retain the carbon skeleton of the compound prior to derivatization.The fluorescent dyes and derivatives thereof listed above can be synthesized or are commercially available.

[0098] Fluorescent dyes having a maximum excitation wavelength in the range of 400 nm to 1200 nm are suitable for optical imaging, and fluorescent dyes having a maximum excitation wavelength (which may also be the maximum absorption wavelength) in the near-infrared range of 650 nm to 900 nm are preferred, as they enable fluorescent imaging of deep tissues of interest. Fluorescent dyes such as fluorescein and its derivatives, rhodamine and its derivatives, BODIPY® and its derivatives, and cyanine and its derivatives can be advantageously selected from molecules having fluorescence emission wavelengths in the range of 400 to 900 nanometers, particularly 500 to 900 nanometers, and more particularly 600 to 900 nanometers.

[0099] Specific fluorescent dyes are the following compounds:

[0100]

[0101] In some embodiments, the diagnostic agent is a fluorescent probe for detecting MMP12, which comprises a peptide having a site cleavable by MMP12 and a fluorophore bound to the peptide. The peptide having a site cleavable by MMP12 and the fluorophore may be bound directly or via a linker. The peptide having a site cleavable by MMP12 can be synthesized by known methods. The fluorophore can be formed by binding any of the fluorescent dyes listed above to the middle or end of the amino acid sequence of the peptide.

[0102] In some embodiments, the diagnostic agent is a fluorescent probe for detecting MMP12, which comprises a conjugate comprising a peptide having a site cleavable by MMP12, a fluorophore attached to one end of the peptide, and a quencher attached to the other end of the peptide. The peptide having a site cleavable by MMP12 and the quencher may be bonded directly or via a linker. Examples of linkers include, but are not limited to, saturated hydrocarbon groups having 5 to 12 carbon atoms or 3 to 5 amino acids, which may have a substituent. The quencher can be formed by binding a quencher compound to the middle or end of the amino acid sequence of the peptide.

[0103] The combination of a fluorophore and a quencher can be any combination known in the art, utilizing the principle of fluorescence control by fluorescence resonance energy transfer (FRET), in which when both are present in a fluorescent probe molecule, the quencher suppresses the fluorescence of the fluorophore, causing the fluorescence to disappear, but the fluorescence increases upon cleavage of the amino acid sequence of the peptide.

[0104] Examples of fluorophores include those formed by binding the fluorescent dye of the above-mentioned labeling substance to a peptide, and preferred examples include fluorescein and its derivatives, cyanine and its derivatives, rhodamine and its derivatives, BODIPY (registered trademark) and its derivatives, coumarin and its derivatives, anthracene and its derivatives, and compounds containing aminobenzyl.

[0105] A quencher is a compound or a part thereof that, when spatially close to a fluorophore, causes the fluorescence of the fluorescent dye to disappear due to energy transfer, stacking, or the like.

[0106] Examples of the quenching compound include compounds in which phenyl groups are bonded to the nitrogen atoms at the 3- and 6-positions of a xanthene ring (e.g., QSY series compounds from ThermoFisher Scientific, etc.), compounds in which aromatic rings are bonded to the nitrogen atoms at the 3- and 6-positions of a xanthene ring of a rhodamine compound (SiR dye) in which the oxygen atoms of rhodamine or a derivative thereof are substituted with silicon atoms, thereby making the compound non-fluorescent and having a fluorescence quantum yield of 0.001 or less (e.g., SinQ series compounds invented by Dr. Kenjiro Hanaoka, one of the inventors; see J. Am. Chem. Soc., 137, 4759-4765 (2015)), etc. (e.g., Black Hole Quencher (BHQ) series compounds from Sigma-Aldrich, etc.), and compounds containing dinitrophenol.

[0107] Specific quenching compounds are the following compounds:

[0108]

[0109] The fluorescent dye and quencher compound can be bound to a peptide having a site cleavable by MMP12 by reacting the functional groups (e.g., carboxyl group, amino group) of the fluorescent dye and quencher compound with the functional group of the terminal amino acid of the peptide, or by forming a covalent bond via a linker, using known methods. The cleavage efficiency of the peptide by MMP can be analyzed by high-performance liquid chromatography. An amino acid sequence with high cleavage activity and selectivity is determined and completed as a probe.

[0110] In some embodiments, the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis.

[0111] According to a fourth aspect of the present invention, there is provided a diagnostic or detection kit for diagnosing or detecting an immune-mediated inflammatory disease characterized by increased expression of MMP12, the diagnostic or detection kit comprising a substance that specifically interacts with MMP12.

[0112] The substance that specifically interacts with MMP12 can be the substance that specifically interacts with MMP12 described in relation to the diagnostic agent of the third embodiment. The substance that specifically interacts with MMP12 may be bound to the labeling substance described in relation to the first or third embodiment. Alternatively, the diagnostic agent of the third embodiment may be used as the substance that specifically interacts with MMP12.

[0113] In addition to the substance that specifically interacts with MMP12, the diagnostic or detection kit may contain various other substances necessary for the reaction to detect the level of MMP12, which do not adversely affect the reaction when stored in the presence of other substances, such as a color-developing reagent, a labeled secondary antibody, and a blocking agent. The diagnostic or detection kit may further contain a buffer solution, a washing solution, instructions for use, etc.

[0114] The substance that specifically interacts with MMP12 may be provided in a state immobilized on a suitable solid support. Examples of solid supports include various carriers made of insoluble polysaccharides (e.g., agarose, dextran, cellulose, etc.), synthetic resins, glass, metals, etc., which are commonly used in antigen-antibody reactions, such as microplates, tubes, membranes, columns, beads, and sensor chips. Immobilization may be performed by physical adsorption or by chemical bonding, which is commonly used to insolubilize and immobilize proteins.

[0115] The diagnostic or detection kit of the present invention can be applied to clinical tests such as the diagnosis, detection, or monitoring of immune-mediated inflammatory diseases characterized by increased expression of MMP12, and can also be used in drug discovery research, etc.

[0116] In some embodiments, the substance that specifically interacts with MMP12 is a substance that binds to MMP12 selected from the group consisting of an antibody against MMP12, an antigen-binding fragment of an antibody against MMP12, and an aptamer against MMP 12. A diagnostic or detection kit comprising such a substance that binds to MMP12 can be used as an in vitro diagnostic or detection kit.

[0117] In some embodiments, the substance that specifically interacts with MMP12 is a substance that is cleaved by MMP12 due to the protease activity of MMP12, for example, a peptide having a site that can be cleaved by MMP12. The substance that is cleaved by MMP12 may be bound to a labeling substance described in the third aspect, or may have a fluorophore and a quencher bound to it. A diagnostic or detection kit comprising such a substance that is cleaved by MMP12 due to the protease activity of MMP12 can be used as an in vivo diagnostic or detection kit.

[0118] According to a fifth aspect of the present invention, there is provided a method for screening for a substance effective in treating an immune-mediated inflammatory disease characterized by increased expression of MMP12, the method comprising measuring the level of MMP12 in a biological sample obtained from a subject after administration of a test substance, and selecting the test substance as a candidate substance effective in treating an immune-mediated inflammatory disease characterized by increased expression of MMP12 if the level of MMP12 is reduced by administration of the test substance.

[0119] The biological sample expressing MMP12 may be a body fluid, tissue, or cell. Preferably, the subject is a human suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12 or a non-human mammalian model of an immune-mediated inflammatory disease characterized by increased expression of MMP12. The level of MMP12 can be measured using a method similar to that described in relation to the first aspect of the present invention.

[0120] The level of MMP12 in a biological sample obtained from a subject administered a test substance is compared with a control value, and if the former is lower than the latter, it is determined that the test substance has reduced MMP12 in the biological sample, and the test substance can be selected as a candidate substance effective in treating an immune-mediated inflammatory disease characterized by increased expression of MMP12.

[0121] The control value may be the level of MMP12 in a biological sample obtained from the same subject before administration of the test substance, the average or median value calculated from measurements of MMP12 levels in multiple healthy subjects, a cutoff value separating a group of healthy subjects from a group of patients diagnosed with an immune-mediated inflammatory disease characterized by increased MMP12 expression, or a certain value that is higher than the average or median value calculated from measurements of MMP12 levels in multiple healthy subjects and lower than the average or median value calculated from measurements of MMP12 levels in multiple patients suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression.

[0122] According to a sixth aspect of the present invention, there is provided a therapeutic agent for immune-mediated inflammatory diseases characterized by increased expression of MMP12, comprising an MMP12 inhibitory substance.

[0123] MMP12 has the ability to degrade extracellular matrices such as collagen, elastin, fibronectin, fibrin, fibrinogen, and proteoglycans, causing rupture of connective tissues such as the elastic lamina in blood vessel walls and glomeruli.

[0124] The MMP12 inhibitor refers to a substance that binds to MMP12 and inhibits the substrate degradation activity of MMP12 and subsequent tissue damage. The same substances as those described for the "substance that binds to MMP12" in the method of the first aspect can be used. The MMP12 inhibitor can be contained as an active ingredient in a therapeutic agent. The MMP12 inhibitor may be a commercially available MMP12 inhibitor or an MMP12 inhibitor obtained or produced by a known method described in the literature. Zinc is required for activation of MMP12. In some embodiments, the MMP12 inhibitor is an MMP12 inhibitor that inhibits zinc binding to the active center region of MMP12 or zinc-mediated catalytic activity of MMP12. Examples of MMP12 inhibitors include, but are not limited to, selective MMP12 inhibitors such as MMP408 (CAS No. 1258003-93-8) and RXP470.1 (CAS No. 891198-31-5).

[0125] In some embodiments, the MMP12 inhibitor can be selected from the group consisting of an antibody against MMP12 (anti-MMP12 antibody), an antigen-binding fragment of an anti-MMP12 antibody, and an aptamer against MMP12.

[0126] The route of administration of the therapeutic agent for immune-mediated inflammatory diseases characterized by increased MMP12 expression, which contains the MMP12 inhibitor of this embodiment, is not particularly limited, and may be an oral or parenteral formulation. For example, the therapeutic agent may be in the form of an oral formulation such as a tablet, capsule, granule, powder, or syrup. It may also be in the form of a parenteral formulation such as an injection, eye drops, nasal drops, ointment, cream, lotion, gel, or spray. These formulations can be produced by known methods.

[0127] For example, when preparing a formulation for oral administration, it can be produced by formulating an appropriate combination of solubilizers such as gum tragacanth, gum arabic, sucrose fatty acid esters, lecithin, olive oil, soybean oil, PEG 400, etc.; excipients such as starch, mannitol, lactose, etc.; binders such as methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, etc.; disintegrants such as crystalline cellulose, calcium carboxymethylcellulose, etc.; lubricants such as talc, magnesium stearate, etc.; and flowability improvers such as light anhydrous silicic acid, etc.

[0128] A typical example of a parenteral formulation is an injection. Injectable formulations can be prepared, for example, by dissolving or diluting an MMP12 inhibitor, such as an anti-MMP12 antibody or an aptamer against MMP12, in physiological saline or a buffer solution for intravenous injection. To increase the solubility of an MMP12 inhibitor, known techniques can be used, such as changing the solvent, changing the salt contained in the solution, changing the salt strength, or encapsulating the MMP12 inhibitor in cyclodextrins. Subcutaneous, intramuscular, and intravenous injections can be prepared by adding a pH adjuster, buffer, stabilizer, isotonicity agent, local anesthetic, etc. to the MMP12 inhibitor using conventional methods. Eye drops may be aqueous eye drops, non-aqueous eye drops, suspension eye drops, emulsion eye drops, eye ointments, etc. Such formulations can be prepared by methods known to those skilled in the art, incorporating pharmaceutically acceptable carriers, particularly those acceptable for eye drops, as needed, as a composition suitable for the administration form, such as isotonicity agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc. Eye drops can be prepared by dissolving or suspending the MMP12 inhibitor in an aqueous solvent such as sterilized purified water or saline, or in a non-aqueous solvent such as a vegetable oil such as cottonseed oil, soybean oil, sesame oil, or peanut oil, adjusting the osmotic pressure to a predetermined level, and subjecting the solution to sterilization, such as filtration sterilization. Commonly used additives, such as isotonicity agents, buffers, and preservatives, are appropriately incorporated into the aqueous base for eye drops. For example, isotonic agents include sodium chloride, potassium chloride, polyhydric alcohols, sugars, etc.; buffers include sodium borate, sodium citrate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.; preservatives include benzethonium hydrochloride, benzalkonium hydrochloride, chlorobutanol, etc. In addition, stabilizers such as glycerin or polysorbate 80 and pH adjusters may be added as needed. Nasal drops and sprays are prepared as liquid formulations containing an MMP12 inhibitor. Nasal drops are preferably placed in a container suitable for application to the nasal cavity. Sprays are prepared by placing the liquid formulation in a spray container containing a propellant. The propellant is a gas such as carbon dioxide.The spray agent may be applied not only to the epidermis but also to the nasal cavity or oral cavity, and a spray container having a shape suited to the mode of use is appropriately selected for preparation.

[0129] The effective dose of an MMP12 inhibitor is adjusted as appropriate depending on various factors, such as the patient's condition and symptoms. It can usually be determined as appropriate from the range of 0.001 to 10 mg / kg / day, preferably 0.01 to 1 mg / kg / day. However, this can be adjusted depending on the administration form, such as using a high dose for systemic administration and a low dose for local administration. When administered orally, the daily dose of an MMP12 inhibitor can be administered once a day or in divided doses. Conversely, a dose equivalent to several days can be administered in one dose, resulting in an administration cycle of once every two or more days. When administered systemically as an injection, the dose of an MMP12 inhibitor can also be administered once a day or in divided doses. Conversely, a dose equivalent to several days can be administered in one injection, resulting in an administration cycle of once every two or more days. Continuous administration by infusion, etc. is also possible. When parenteral preparations such as eye drops, nasal drops, ointments, lotions, creams, gels, and sprays are administered locally, the amount of MMP12 inhibitor in the topical treatment, the frequency of topical administration, and the area of ​​application can be adjusted as appropriate. In either case, administration does not necessarily have to be continuous or regular, and can be carried out at appropriate intervals depending on changes in symptoms, etc. If a single administration results in cure or remission, there is no need to administer multiple doses. Administration can be resumed if symptoms recur or worsen.

[0130] The method of administration of the therapeutic agent of this embodiment is not particularly limited. However, since vasculitis syndrome, a typical example of a disease to be treated, causes inflammation in blood vessels, systemic administration via intravenous injection or infusion is preferred. When an anti-MMP12 antibody or aptamer is used as the MMP12 inhibitor, parenteral administration is generally selected because it is considered difficult to deliver a therapeutically effective amount to the affected area via oral administration. The administration method is not limited to intravenous injection or infusion, and local administration such as intramuscular injection may also be used. The administration period can be adjusted appropriately depending on the patient's condition. The administration dose during the administration period can be adjusted appropriately, but examples of administration forms include continuous administration of a fixed amount, or administration of a relatively high dose only at the beginning of administration followed by a lower maintenance dose.

[0131] The therapeutic agent containing the MMP12 inhibitor of this embodiment can also be used in combination with other additives or components that contribute independently to the treatment of immune-mediated inflammatory diseases characterized by increased expression of MMP12. The components used in combination with the MMP12 inhibitor can be administered simultaneously with the MMP12 inhibitor or at different times, and the administration routes for both can be the same or different.

[0132] The components used in combination can be purchased commercially or obtained or prepared by methods described in the literature. The amount of each component to be applied can also be determined appropriately based on publicly known information.

[0133] The present invention encompasses the following embodiments: Item 1. A method for detecting an immune-mediated inflammatory disease characterized by increased expression of MMP12 in a subject, the method comprising measuring the level of MMP12 in a biological sample obtained from the subject. Item 2. The method of Item 1, wherein measuring the level of MMP12 comprises measuring the protein level of MMP12. Item 3. The method of Item 1, further comprising contacting the biological sample obtained from the subject with a substance that binds to MMP12, and measuring the level of MMP12 comprises measuring the level of a complex between MMP12 and the substance that binds to MMP12. Item 4. The method of Item 1, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitic syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis. Item 5. The method of Item 1, wherein the biological sample is blood, serum, or plasma. Item 6. Item 7. The method of Item 1, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is Takayasu's arteritis. Item 8. The method of Item 1, further comprising contacting a biological sample obtained from the subject with a peptide having a site cleavable by MMP12, and measuring the level of MMP12 comprises measuring a change caused by cleavage of the peptide having a site cleavable by MMP12. Item 9. The method of any one of Items 1 to 7, wherein the method is for predicting the likelihood of developing an immune-mediated inflammatory disease characterized by increased MMP12 expression in a subject, and further comprises comparing the level of MMP12 in the biological sample obtained from the subject with a reference value, and wherein a higher level of MMP12 in the biological sample obtained from the subject compared to the reference value indicates a high likelihood that the subject will develop an immune-mediated inflammatory disease. The method is a method for predicting the prognosis of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the subject has been treated for the immune-mediated inflammatory disease characterized by increased expression of MMP12,Item 10. The method of any one of Items 1 to 7, further comprising comparing the level of MMP12 in the biological sample obtained from the subject with a reference value, wherein a higher level of MMP12 in the biological sample obtained from the subject as compared to the reference value indicates a high likelihood of relapse of the immune-mediated inflammatory disease characterized by increased MMP12 expression in the subject. Item 10. The method of any one of Items 1 to 7, further comprising comparing the level of MMP12 in the biological sample obtained from the subject with a reference value, wherein a lower level of MMP12 in the biological sample obtained from the subject as compared to the reference value indicates a high likelihood of remission of the immune-mediated inflammatory disease characterized by increased MMP12 expression in the subject. Item 12. The method according to any one of Items 1 to 7, wherein the method is for evaluating the timing of administration of a therapeutic agent for an immune-mediated inflammatory disease characterized by increased MMP12 expression to a subject suffering from an immune-mediated inflammatory disease characterized by increased MMP12 expression, and the method further comprises comparing the level of MMP12 in a biological sample obtained from the subject with a reference value, and a lower level of MMP12 in the biological sample obtained from the subject compared to the reference value indicates cessation of administration of the agent to the subject. Item 13. A biomarker for diagnosing an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising MMP12. Item 13. The biomarker of Item 12, comprising MMP12 protein. Item 14. A diagnostic agent for an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising a substance that specifically interacts with MMP12. Item 15. Item 16. The diagnostic agent according to Item 14, which is an in vivo diagnostic agent comprising a conjugate of a peptide having a site cleavable by MMP12 and a labeling substance. Item 17. The substance that specifically interacts with MMP12 is a peptide having a site cleavable by MMP12,Item 16. The diagnostic agent according to Item 15, wherein the conjugate comprises a peptide having a site cleavable by MMP12, a fluorophore bound to one end of the peptide, and a quencher bound to the other end of the peptide. Item 17. The diagnostic agent according to Item 15, wherein the labeling substance comprises a fluorescent dye having a fluorophore, and the conjugate comprises a peptide having a site cleavable by MMP12, a fluorophore bound to one end of the peptide, and a quencher bound to the other end of the peptide. Item 18. Item 18. The diagnostic agent according to any one of Items 14 to 17, wherein the peptide having a site cleavable by MMP12 is either (i) or (ii) below: (i) human CXCL1 (UniPlot P09341), human CXCL2 (UniPlot P19875), human CXCL3 (UniPlot P19876), human CXCL5 (UniPlot P42830), human CXCL8 (UniPlot P10145), mouse CXCL1 (UniPlot A2RTH0), mouse CXCL2 (UniPlot P12850), mouse CXCL3 (UniPlot P10889), mouse CXCL5 (UniPlot Item 19. A peptide having a total length of 8 to 25 amino acids, which is a partial sequence of any of the amino acid sequences of ELRCXC (X is Q or V), at least 1 to 5 amino acids upstream thereof, and one or more amino acids downstream thereof, and which is a peptide having an amino acid sequence of 8 to 25 amino acids in total. (ii) A peptide of the above (i), in which one or more amino acids upstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted, one or more amino acids downstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted, or one or more amino acids upstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted and one or more amino acids downstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted. Item 18. The diagnostic agent according to any one of Items 14 to 17, wherein the peptide having a site cleavable by MMP12 is any one of the following (i) to (vi): (i) a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 9, (ii) a peptide consisting only of an amino acid sequence of any one of SEQ ID NOs: 1 to 9, or (iii)Item 20. A peptide consisting of a partial sequence of a peptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 9, which has the portion ELRCXC (X is Q or V), (iv) a peptide of any one of (i) to (iii) above, in which one or more of the amino acids at positions 1 to 5 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 have been substituted, one or more of the amino acids at positions 12 to 13 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 have been substituted, or both one or more of the amino acids at positions 1 to 5 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 and one or more of the amino acids at positions 12 to 13 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 have been substituted. Item 21. The diagnostic agent according to any one of Items 14 to 17, wherein the peptide having a site cleavable by MMP12 is any one of the following (i) to (v): (i) a peptide having an amino acid sequence of any one of SEQ ID NOs: 10 to 32, (ii) a peptide consisting only of an amino acid sequence of any one of SEQ ID NOs: 10 to 32, (iii) a peptide consisting of a partial sequence of a peptide having an amino acid sequence of any one of SEQ ID NOs: 10 to 32, having a site cleavable by MMP12, and having a total length of 8 to 25 amino acids, (iv) a peptide in which one or two amino acids other than the amino acid having the site cleavable by MMP12 are substituted in any one of the peptides (i) to (iii) above, or (v) a peptide in which all cysteines in the native sequence are substituted with other amino acids such as serine in any one of the peptides (i) to (iii) above. Item 18. The diagnostic agent according to any one of Items 14 to 17, wherein the peptide having a site cleavable by MMP12 is any one of the following (i) to (v): (i) a peptide having an amino acid sequence of any one of SEQ ID NOs: 11, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31; (ii) a peptide consisting of only an amino acid sequence of any one of SEQ ID NOs: 111, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31;(iii) a peptide consisting of a partial sequence of a peptide having any of the amino acid sequences of SEQ ID NOs: 111, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31, which has a site cleavable by MMP12, and which has a total length of 8 to 25 amino acids, (iv) a peptide in any of the peptides (i) to (iii) above, in which one or two amino acids other than the amino acid having the site cleavable by MMP12 have been substituted, (v) a peptide in any of the peptides (i) to (iii) above, in which all cysteines in the native sequence have been substituted with other amino acids such as serine. Item 22. The diagnostic agent according to any one of Items 14 to 21, wherein the peptide having a site cleavable by MMP12 is a peptide consisting of a sequence of 8 to 25 amino acids. Item 23. The diagnostic agent according to any one of Items 14 to 21, wherein the peptide having a site cleavable by MMP12 is a peptide consisting of a sequence of 10 to 20 amino acids. Item 24. Item 24. The diagnostic agent according to any one of Items 14 to 21, wherein the peptide having a site cleavable by MMP12 is a peptide consisting of a sequence of 12 to 18 amino acids.the labeling substance comprises a fluorescent dye having a fluorophore, and the fluorescent dye is, for example, phenyl and derivatives thereof, naphthalene and derivatives thereof such as 5-dimethylaminonaphthalene-1-sulfonic acid and hydroxynaphthalenes, anthracene and derivatives thereof such as 9,10-diphenylnaphthalene and 9-methylanthracene, pyrene and derivatives thereof such as N-(1-pyrene)iodoacetamide and hydroxypyrenes, biphenyl and derivatives thereof, acridine and derivatives thereof such as hydroxyacridines and 9-methylacridine, coumarin and derivatives thereof such as 7-dialkylamino-4-methylcoumarin and 4-bromomethyl-7-methoxycoumarin, xanthene and derivatives thereof, phthalocyanine and derivatives thereof, stilbene and derivatives thereof such as 6,6'-dibromostilbene and hydroxystilbenes, furan and derivatives thereof, oxazole and derivatives thereof, oxadiazole and derivatives thereof, nitrobenzoxadiazole and derivatives thereof such as hydroxynitrobenzoxadiazoles, benzothiazole and derivatives thereof, fluorescein and 5-iodoacetamide midofluorescein and derivatives thereof such as fluorescein-5-maleimide, rhodamine and derivatives thereof such as tetramethylrhodamine, tetraethylrhodamine, carboxytetramethylrhodamine, BODIPY (registered trademark) and derivatives thereof, eosin and derivatives thereof, erythrosine and derivatives thereof such as hydroxyerythrosines and 5-iodoacetamidoerythrosine, resorufin and derivatives thereof such as hydroxyresorufin, quinoline and derivatives thereof such as 6-hydroxyquinoline and 6-aminoquinoline, carbazole Item 25. The diagnostic agent according to Item 15 or 16, wherein the fluorescent dye is at least one selected from the group consisting of benzoindol and derivatives thereof such as N-methylcarbazole, cyanine and derivatives thereof such as hydroxycyanine, carbocyanine and derivatives thereof such as phenylcarbocyanine, pyridinium salts and derivatives thereof such as 4-(4-dialkyldiamidostyryl)-N-methylpyridinium iodate, fluorescent complexes of lanthanides and derivatives thereof, green fluorescent protein (GFP) and derivatives thereof, and benzoindole and derivatives thereof (such as indocyanine green).Item 27. The diagnostic agent according to any one of Items 14 to 26, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis. Item 28. A diagnostic kit for diagnosing an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising a substance that specifically interacts with MMP12. Item 29. Item 30. The diagnostic kit according to Item 28, wherein the substance that specifically interacts with MMP12 is a substance that binds to MMP12 selected from the group consisting of an antibody against MMP12, an antigen-binding fragment of an antibody against MMP12, and an aptamer against MMP12. Item 31. The diagnostic kit according to Item 28, wherein the substance that specifically interacts with MMP12 comprises a peptide having a site cleavable by MMP12. Item 32. The diagnostic kit according to Item 26, wherein the peptide having a site cleavable by MMP12 has a labeling substance bound to it. Item 33. The diagnostic kit according to Item 26, wherein a fluorophore is bound to one end of the peptide having a site cleavable by MMP12, and a quencher is bound to the other end of the peptide. Item 34. A diagnostic kit according to any one of Items 30 to 32, wherein the peptide having a site cleavable by MMP12 is either (i) or (ii) below: (i) human CXCL1 (UniPlot P09341), human CXCL2 (UniPlot P19875), human CXCL3 (UniPlot P19876), human CXCL5 (UniPlot P42830), human CXCL8 (UniPlot P10145), mouse CXCL1 (UniPlot A2RTH0), mouse CXCL2 (UniPlot P12850), mouse CXCL3 (UniPlot P10889), mouse CXCL5 (UniPlotItem 33. A peptide having a total length of 8 to 25 amino acids, which is a partial sequence of any of the amino acid sequences of ELRCXC (X is Q or V), at least one to five amino acid sequences upstream thereof, and one or more amino acid sequences downstream thereof, and which is a peptide having a total length of 8 to 25 amino acids. (ii) A peptide of the above (i), in which one or more amino acids upstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted, one or more amino acids downstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted, or one or more amino acids upstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted and one or more amino acids downstream of the amino acid sequence of ELRCXC (X is Q or V) have been substituted. Item 34. A diagnostic kit according to any one of Items 30 to 32, wherein the peptide having a site cleavable by MMP12 is any one of the following (i) to (vi): (i) a peptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 9, (ii) a peptide consisting only of the amino acid sequence of any one of SEQ ID NOs: 1 to 9, (iii) a peptide consisting of a partial sequence of a peptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 9, which has the portion ELRCXC (X is Q or V), or (iv) a peptide of any one of (i) to (iii) above, wherein one or more of the amino acids at positions 1 to 5 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 have been substituted, or one or more of the amino acids at positions 12 to 13 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 have been substituted, or both one or more of the amino acids at positions 1 to 5 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 and one or more of the amino acids at positions 12 to 13 of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 have been substituted. Item 33. The diagnostic kit according to any one of Items 30 to 32, wherein the peptide having a site cleavable by MMP12 is any one of the following (i) to (v): (i) a peptide having an amino acid sequence of any one of SEQ ID NOs: 10 to 32; (ii) a peptide consisting only of an amino acid sequence of any one of SEQ ID NOs: 10 to 32;(iii) a peptide consisting of a partial sequence of a peptide having an amino acid sequence of any one of SEQ ID NOS: 10 to 32, which has a site cleaved by MMP12, and which has a total length of 8 to 25 amino acids, (iv) a peptide in which one or two amino acids other than the amino acid having the site cleaved by MMP12 are substituted in any one of the peptides (i) to (iii) above, (v) a peptide in which all cysteines in the native sequence are substituted with other amino acids such as serine in any one of the peptides (i) to (iii) above. Item 33. The diagnostic kit according to any one of Items 30 to 32, wherein the peptide having a site cleavable by MMP12 is any one of the following (i) to (v): (i) a peptide having an amino acid sequence of any one of SEQ ID NOs: 11, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31; (ii) a peptide consisting of only an amino acid sequence of any one of SEQ ID NOs: 111, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31; (iii) a peptide consisting of a partial sequence of a peptide having an amino acid sequence of any one of SEQ ID NOs: 111, 13, 14, 15, 16, 20, 24, 25, 26, 27, 29, and 31 having a site cleavable by MMP12, the peptide consisting of a total length of 8 to 25 amino acids; (iv) a peptide in which one or two of the amino acids other than the amino acid having the site cleavable by MMP12 are substituted in any of the peptides (i) to (iii) above, (v) a peptide in which all of the cysteines in the native sequence are substituted with other amino acids such as serine in any of the peptides (i) to (iii) above. Item 37. The diagnostic kit according to any one of Items 30 to 37, wherein the peptide having the site cleavable by MMP12 is a peptide consisting of a sequence of 8 to 25 amino acids. Item 38. The diagnostic kit according to any one of Items 30 to 37, wherein the peptide having the site cleavable by MMP12 is a peptide consisting of a sequence of 10 to 20 amino acids. Item 39. The diagnostic kit according to any one of Items 30 to 37, wherein the peptide having the site cleavable by MMP12 is a peptide consisting of a sequence of 12 to 18 amino acids. Item 40.The labeling substance includes a fluorescent dye having a fluorophore, and the fluorescent dye is preferably phenyl and derivatives thereof, naphthalene and derivatives thereof such as 5-dimethylaminonaphthalene-1-sulfonic acid and hydroxynaphthalenes, anthracene and derivatives thereof such as 9,10-diphenylnaphthalene and 9-methylanthracene, pyrene and derivatives thereof such as N-(1-pyrene)iodoacetamide and hydroxypyrenes, biphenyl and derivatives thereof, acridine and derivatives thereof such as hydroxyacridines and 9-methylacridine, coumarin and derivatives thereof such as 7-dialkylamino-4-methylcoumarin and 4-bromomethyl-7-methoxycoumarin, xanthene and derivatives thereof, phthalocyanine and derivatives thereof, stilbene and derivatives thereof such as 6,6'-dibromostilbene and hydroxystilbenes, furan and derivatives thereof, oxazole and derivatives thereof, oxadiazole and derivatives thereof, nitrobenzoxadiazole and derivatives thereof such as hydroxynitrobenzoxadiazoles, benzothiazole and derivatives thereof, fluorescein and 5-iodoacetamide Item 32. The diagnostic kit according to Item 31, wherein the fluorescent dye is at least one selected from amidofluorescein and derivatives thereof such as fluorescein-5-maleimide, rhodamine and derivatives thereof such as tetramethylrhodamine, tetraethylrhodamine, and carboxytetramethylrhodamine, BODIPY (registered trademark) and derivatives thereof, eosin and derivatives thereof, erythrosine and derivatives thereof such as hydroxyerythrosines and 5-iodoacetamidoerythrosine, resorufin and derivatives thereof such as hydroxyresorufin, quinoline and derivatives thereof such as 6-hydroxyquinoline and 6-aminoquinoline, derivatives thereof such as carbazole and N-methylcarbazole, derivatives thereof such as cyanine and hydroxycyanine, derivatives thereof such as carbocyanine and phenylcarbocyanine, pyridinium salts and derivatives thereof such as 4-(4-dialkyldiamidostyryl)-N-methylpyridinium iodate, fluorescent complexes of lanthanides and derivatives thereof, green fluorescent protein (GFP) and derivatives thereof, and benzoindole and derivatives thereof (such as indocyanine green). Section 41.Item 42. The diagnostic kit according to any one of Items 28 to 41, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis. Item 43. A method for diagnosing an immune-mediated inflammatory disease characterized by increased MMP12 expression in a subject, using the diagnostic agent according to any one of Items 14 to 27 or the diagnostic kit according to any one of Items 28 to 42. Item 44. 44. A method for screening for a substance effective in treating an immune-mediated inflammatory disease characterized by increased MMP12 expression, the method comprising: measuring the level of MMP12 in a biological sample obtained from a subject after administration of a test substance; and selecting the test substance as a candidate substance effective in treating an immune-mediated inflammatory disease characterized by increased MMP12 expression if the administration of the test substance reduces the MMP12 level. 45. The method of 44, wherein measuring the level of MMP12 comprises measuring the level of a complex between MMP12 and a substance that binds to MMP12. 46. The method of 45, wherein the substance that binds to MMP12 is selected from the group consisting of an antibody against MMP12, an antigen-binding fragment of an antibody against MMP12, and an aptamer against MMP12. 47. Item 44. The method according to Items 44 to 46, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and non-tuberculous mycobacteriosis. Item 48. The method according to Items 44 to 47, wherein the biological sample is blood, serum, or plasma. Item 49. A therapeutic agent for an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising an MMP12 inhibitor. Item 50.The therapeutic agent according to Paragraph 49, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis. Paragraph 51. The therapeutic agent according to Paragraph 49, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression includes ANCA-associated vasculitis. Paragraph 52. A method for treating an immune-mediated inflammatory disease in a subject in need of treatment for an immune-mediated inflammatory disease characterized by increased MMP12 expression, comprising administering a therapeutically effective amount of an MMP12 inhibitor to the subject. Paragraph 53. The method according to Paragraph 52, wherein the immune-mediated inflammatory disease characterized by increased MMP12 expression is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and nontuberculous mycobacteriosis. Item 54. The method according to Item 52, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 comprises vasculitis or granulomatous vasculitis.

[0134] The disclosures of all patent applications and literature cited in this specification are hereby incorporated by reference in their entirety. The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0135] Example 1: Identification of vasculitis-specific molecules To screen for vasculitis-specific molecules using serum proteome, we used the Inflammation panel, Immune response panel, Cardiovascular II panel, and Cardiovascular III panel of the Olink proximity extension assay (Uppsala), which involves binding two antibodies with oligo-DNA conjugated to a single target antigen protein and detecting and quantifying the double-stranded DNA sequence obtained by hybridization using qPCR. Of the 368 proteins measured, 354 proteins, excluding duplicates, were evaluated. Patients included those with giant cell arteritis (GCA), Takayasu arteritis (TAK), polyarteritis nodosa (PAN), granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA), microscopic polyangiitis (MPA), Behcet's disease (BD), relapsing polychondritis (RP), adult-onset Still's disease (AOSD), and healthy controls. Based on clinical characteristics, patients were classified into five groups (1: GCA, TAK, PAN, GPA, EGPA, MPA; 2: AOSD, BD, RP; 3: PMR, RA, SpA; 4: SLE, SSc, IIM; 5: IgG4RD, SjS). Multiple correlation tests identified proteins that were significantly elevated in each group compared with healthy controls. The identified proteins were plotted on a Venn diagram to identify molecules specific to vasculitis (group 1). Results: 6 molecules were identified for group 1; 17 for group 2; 0 for group 3; 9 for group 4; and 1 for group 5.We performed receiver operating characteristic (ROC) analysis using NPX values ​​measured by Olink to compare vasculitis syndrome patients with healthy controls and patients with immune-mediated inflammatory diseases other than vasculitis syndromes, focusing on molecules 1-6 specific to vasculitis (group 1). We found that MMP12 (solid line) had the highest sensitivity for determining AUC (Figure 1(A) and 1(B)). Validation using serum samples by ELISA (Thermo Fisher Scientific) revealed that serum MMP12 levels were characteristically elevated in patients with granulomatous vasculitis (GCA, EGPA, GPA, MPA, PAN, and TAK), as well as in patients with IgG4-related disease (IgG4RD), inflammatory bowel disease, and nontuberculous mycobacterial disease (NTM) (Figure 2). Serum MMP12 levels were also elevated in some patients with polymyalgia rheumatica (PMR), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). In the control immune-mediated inflammatory diseases, no increase in MMP12 expression was observed, making it useful for differentiating these diseases. Furthermore, MMP12 levels were normal in patients with common bacterial and viral infections, making MMP12 useful for distinguishing these diseases from vasculitis syndromes.

[0136] Example 2: Expression of MMP12 in Tissues. MMP12 is not expressed in peripheral blood immune cells. To clarify whether MMP12 originates from certain tissues, immunohistochemical staining for MMP12 was performed on vasculitis tissues from Takayasu's arteritis (TAK), giant cell arteritis (GCA), and granulomatosis with polyangiitis (GPA), immune-mediated inflammatory diseases characterized by increased MMP12 expression. MMP12 was stained using goat anti-human MMP12 (R&D) and compared with HE staining of serial sections and immunohistochemical staining for CD68 and CD206. (Results) Immunohistochemical staining revealed MMP12 expression in the aorta of a patient with Takayasu's arteritis (Figure 3), the temporal artery of a patient with giant cell arteritis (Figure 4), and the lung of a patient with granulomatosis with polyangiitis (Figure 5). Comparison with HE staining revealed that MMP12 was expressed in histiocytes and multinucleated giant cells. Histochemical staining revealed that MMP12-positive cells were CD68- and CD206-positive, confirming that MMP12 originated from vasculitis tissue macrophages. Alveolar macrophages were CD68- and CD206-positive, but MMP12-negative, revealing that a portion of CD206-positive macrophages exhibited an MMP12-positive phenotype.

[0137] Example 3: Correlation with PET-CT score (PETVAS) MMP12 was measured by ELISA (Thermo Fisher Scientific) using serum samples from patients with Takayasu's arteritis and giant cell arteritis before treatment. The PET vascular activity score (PETVAS) used in PET-CT examinations is known to be associated with the relapse rate, which is a prognostic indicator of treatment for Takayasu's arteritis and giant cell arteritis (Arthritis Rheumatol 2018;70:439-449.). Therefore, we investigated the correlation between serum MMP12 and PETVAS in patients who had both serum MMP12 levels and PETVAS. In this example, the PETVAS cutoff was set at 20 or higher, which has been reported to be associated with a high relapse rate. The serum MMP12 cutoff was set at a value (100 pg / ml) that distinguishes between healthy subjects and patients diagnosed by a physician with an immune-mediated inflammatory disease characterized by elevated MMP12 expression, and at a fixed value (500 pg / ml) that is higher than the mean calculated from measurements of MMP12 levels in multiple healthy subjects and lower than the mean calculated from measurements of MMP12 levels in multiple patients with immune-mediated inflammatory diseases characterized by elevated MMP12 expression. (Results) MMP12 levels at the time of diagnosis of Takayasu's arteritis and giant cell arteritis were positively correlated with PETVAS (Figure 6). MMP12 was associated with relapse rate, a prognostic indicator of treatment, regardless of whether PETVAS levels were high (≥20) or low (<20) (Figure 7), demonstrating that high serum MMP12 levels are associated with a poor prognosis. The method of this embodiment may be a better evaluation tool than PETVAS and is useful for stratifying the risk of relapse of subjects.

[0138] Example 4: Evaluation of Remission Using MMP12. MMP12 was measured by ELISA (Thermo Fisher Scientific) using serum samples from patients with Takayasu's arteritis and giant cell arteritis before and after treatment. Serum MMP12 levels were measured serially in patients who maintained remission without relapse (patients 1-19; Takayasu's arteritis: n=9; giant cell arteritis: n=10). The time course of serum MMP12 levels was compared with the time course of imaging tests, such as contrast-enhanced CT, contrast-enhanced MRI, and PET-CT, which are indicators of vasculitis activity. (Results) Among patients 1-19 with Takayasu's arteritis and giant cell arteritis who maintained remission without relapse, patient 1-11 showed a monotonically decreasing pattern in which MMP12 levels normalized with treatment and then maintained normal (Figure 8(A)). Patients 12-19 showed a smoldering pattern in which MMP12 levels did not normalize or normalized and then re-elevated (Figure 9(A)). In the monotonically descending type, disease activity such as vascular wall thickening observed on imaging tests disappeared, and MMP12 was useful as a criterion for assessing remission (Figures 8(B) and (C)). On the other hand, in the smoldering type, vascular wall thickening persisted, reflecting latent disease activity (Figures 9(B) and (C)). The monotonically descending type (n = 11) consisted of Takayasu's arteritis (n = 3) and giant cell arteritis (n = 9), while the smoldering type (n = 8) consisted of Takayasu's arteritis (n = 6) and giant cell arteritis (n = 2). Of the smoldering type Takayasu's arteritis (n = 6), two patients had inflammatory bowel disease (IBD). This suggests that Takayasu's arteritis, especially Takayasu's arteritis complicated by IBD, may have persistently high MMP12 levels and be less likely to achieve remission.

[0139] Example 5: Evaluation of Drug Withdrawal Using MMP12 as an Indicator Among patients 1-19 with Takayasu's arteritis and giant cell arteritis who maintained remission without relapse, patient 19 maintained drug-free remission. Serum samples from patients who maintained drug-free remission were used to measure MMP12 using ELISA (Thermo Fisher Scientific). (Results) After discontinuation of steroids and tocilizumab (Actemra®), a transient increase in serum MMP12 was observed, but subsequently decreased and maintained at a normal MMP12 level (<100 pg / ml). Normal MMP12 may be useful as an indicator of drug withdrawal.

[0140] Example 6: Evaluation of Relapse Under Immunosuppressive Therapy, Including IL-6 Receptor Inhibitors. MMP12 was measured by ELISA (Thermo Fisher Scientific) using serum samples from patients with Takayasu's arteritis and giant cell arteritis before and after treatment. Serial serum MMP12 measurements were plotted for patients who experienced a relapse within 2 years of treatment (patients 20-32; Takayasu's arteritis: n = 4; giant cell arteritis: n = 9). The time course of serum MMP12 levels was compared with the time course of imaging tests, such as contrast-enhanced CT, contrast-enhanced MRI, and PET-CT, which are indicators of vasculitis activity. (Results) Among patients 20-31 with Takayasu's arteritis and giant cell arteritis who experienced a relapse, serum MMP12 levels normalized during the remission phase and increased during the relapse in all patients. Furthermore, in five of the six patients for whom pre-relapse samples were available, elevated MMP12 levels preceded the diagnosis of relapse based on clinical symptoms or imaging tests (Figure 10(A)). The vascular wall thickening that disappeared during the remission period was observed again during the relapse period (FIGS. 10(B)-(D)).

[0141] Among patients with Takayasu's arteritis and giant cell arteritis who experienced a relapse, 7 cases experienced a relapse while using tocilizumab (Actemra®), but MMP12 reflected disease activity before and during the relapse, as did the 5 cases who experienced a relapse while not using tocilizumab (Actemra®).

[0142] Example 7: Therapeutic Effect of MMP12 Inhibitor on ANCA-Associated Vasculitis Model Mice. SCG / ThpNkc mice (see Proc Natl Acad Sci US A. 1993 Apr 15;90(8):3413-7) are a mouse model of ANCA-associated vasculitis generated by crossbreeding two strains, MRL / lpr and BXSB. They are available from the Laboratory Animal Research Resource Bank of the National Institutes of Biomedical Innovation, Health and Nutrition (Resource No. nbio133). In this experiment, male SCG / ThpNkc mice were administered MMP408 (CAS No. 1258003-93-8), a selective MMP12 inhibitor, to examine the therapeutic effect of MMP408. SCG / ThpNkc mice were housed in cages with normal chow and water. Urinary occult blood was confirmed to be positive at 10 weeks of age. Starting from week 11, MMP408 was administered intraperitoneally twice weekly at a dose of 100 μg. Urinalysis was performed at weeks 15 and 20, and mice were sacrificed at week 20. The numbers of mice in the MMP408-treated and non-treated groups were n = 25 and n = 4, respectively. SCG / ThpNkc mice die between weeks 10 and 20, which is similar to the clinical course of ANCA-associated vasculitis, making them suitable for drug screening using survival curves. As shown in Figure 11, 92% of mice in the non-MMP408 treated group died by week 20, while only 2 of 4 mice in the MMP408 treated group survived until week 20 (log-rank test p = 0.037). Furthermore, occult bleeding improved at week 20, as shown in Figure 12.

[0143] Example 8: Development of an MMP12 Detection Probe 1. Peptide Selection We sought substrate peptides highly selective for MMP12 as components of an MMP12 detection probe. To construct the MMP12 detection probe, we first investigated the peptide portion around the cleavage sites of known proteins known to be cleaved by MMP12, and selected candidate peptides No. 1–23 (Figures 13 and 14, Tables 1 and 2). PP-2b and PJ-8 were positive controls, and the arrows in Figures 13 and 14 indicate the cleavage sites by MMP12. The peptides were selected based on the following considerations: 1) The fluorescent dye was introduced at the N-terminus. 2) Glycine (Gly) was introduced as a spacer to reduce the effect of the fluorescent dye on enzyme recognition. 3) Considering the possibility that secondary structure may be involved in MMP12 enzyme recognition, secondary structure was included near the peptide cleavage site. 4) The peptide length was set to approximately 15 amino acid residues to facilitate peptide synthesis.

[0144]

[0145]

[0146] 2. Synthesis of Fluorescently Labeled Peptides First, peptides were synthesized using a Biotage® peptide synthesizer (resin: MBHA, condensing agent: DIC / Oxyma, detergent: DMF, 5 equivalents of amino acids). Next, the peptides were modified with 5-carboxyfluorescein (5-CF) (5-CF: 1.2 equivalents, condensing agent: DIC / Oxyma, detergent: DMF / DCM, dried in a desiccator for 2 hours). Next, the peptide side chains were deprotected, and the peptide was de-resinized (using TFA / Water / TIS, detergent: TFA). The product was crudely purified by ether precipitation, purified by HPLC, and its purity was confirmed by HPLC analysis. The synthesis of the desired peptide was confirmed by mass spectrometry. The synthesized peptides were then subjected to cleavage evaluation as described below.

[0147] 3. Evaluation of Candidate Peptide Cleavage by MMP12 The evaluation of candidate peptide cleavage by MMP12 was performed as follows. On day 1, hMMP12 was diluted to 50 μg / mL in assay buffer. APMA was added to the hMMP12 solution to adjust the final APMA concentration to 1 mM, and the mixture was incubated at 37°C for 24 hours to activate hMMP12. On day 2, the activated hMMP12 was diluted to 2 ng / μL (40 nM) in assay buffer. The substrate (peptide) was diluted to 40 μM in assay buffer. 50 μL each of hMMP12 (2.0 ng / μL) and the substrate (peptide) was mixed and incubated at 37°C for 16 hours. A blank was prepared by mixing equal volumes of assay buffer and substrate (peptide). On the third day, 100 μL of acetonitrile was added to the mixture from day 2, and the mixture was centrifuged for 10 minutes. The supernatant was then collected and analyzed by ultra-performance liquid chromatography (UPLC) at 440 nm with a flow rate of 0.4 mL / min. A: H2O with 0.1% TFA; B: CH3CN with 0.1% TFA. The elution conditions were 10% B → 70% B in 9.5 min. UPLC confirmed that the two positive controls were cleaved by MMP12 (data not shown).

[0148] 4. Evaluation of Cleavage of Each Candidate Peptide by MMP12 Cleavage of candidate peptides Nos. 2, 4-7, 11, 15-17, 19, 20, and 22 by MMP12 was evaluated, as was the positive control. The results are shown in Table 3. Cleavage by MMP12 was confirmed for all tested peptides. Nos. 2, 4-7 have cleavage sites at LQ and LR / K, but the cleavage rates varied. Nos. 2 and 4 had a moderate cleavage rate, while Nos. 5, 6, and 7, which have NLQE, had a moderate to high cleavage rate. Nos. 11, 15, 17, 20, and 22 have cleavage sites at EL, but the cleavage rates varied. Despite having ELI, No. 11 had a high cleavage rate, while No. 15 had a low cleavage rate. Nos. 17, 20, and 22, which have ELR, had high cleavage rates.

[0149]

Claims

1. A method for detecting an immune-mediated inflammatory disease characterized by increased expression of MMP12 in a subject, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitic syndromes, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and non-tuberculous mycobacteriosis, the method comprising measuring the level of MMP12 protein in a biological sample obtained from the subject.

2. contacting the biological sample obtained from the subject with a substance that binds to MMP12; The method of claim 1, wherein measuring the level of MMP12 protein comprises measuring the level of a complex between MMP12 protein and a substance that binds to MMP12 protein.

3. 10. The method of claim 1, wherein the biological sample is blood, serum, or plasma.

4. The method according to claim 1, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is Takayasu's arteritis.

5. contacting the biological sample obtained from the subject with a peptide having a site cleavable by MMP12; The method of claim 1, wherein measuring the level of MMP12 protein comprises measuring a change caused by cleavage of a peptide having a site cleavable by MMP12.

6. The method is a method for predicting the likelihood of developing an immune-mediated inflammatory disease characterized by increased expression of MMP12 in a subject, The method further comprises comparing the level of MMP12 protein in the biological sample obtained from the subject with a reference value; The method according to any one of claims 1 to 4, wherein a high level of MMP12 protein in a biological sample obtained from the subject compared to a reference value indicates that the subject is likely to develop an immune-mediated inflammatory disease.

7. The method is a method for predicting the prognosis of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, the subject has been treated for an immune-mediated inflammatory disease characterized by increased expression of MMP12; The method further comprises comparing the level of MMP12 protein in the biological sample obtained from the subject with a reference value; The method according to any one of claims 1 to 4, wherein a high level of MMP12 protein in a biological sample obtained from the subject compared to a reference value indicates a high likelihood of relapse of an immune-mediated inflammatory disease characterized by increased expression of MMP12 in the subject.

8. The method is a method for predicting a post-treatment condition of a subject suffering from an immune-mediated inflammatory disease characterized by increased expression of MMP12, the subject has been treated for an immune-mediated inflammatory disease characterized by increased expression of MMP12; The method further comprises comparing the level of MMP12 protein in the biological sample obtained from the subject with a reference value; The method according to any one of claims 1 to 4, wherein, when the level of MMP12 protein in a biological sample obtained from the subject is lower compared to a reference value, it indicates that an immune-mediated inflammatory disease characterized by increased expression of MMP12 in the subject is likely to be in remission.

9. The method is a method for evaluating the timing of administration of a therapeutic agent for an immune-mediated inflammatory disease characterized by increased expression of MMP12 to a subject suffering from the immune-mediated inflammatory disease characterized by increased expression of MMP12; The method further comprises comparing the level of MMP12 protein in the biological sample obtained from the subject with a reference value; The method according to any one of claims 1 to 4, wherein a lower level of MMP12 protein in a biological sample obtained from the subject compared to a reference value indicates that administration of the drug to the subject should be discontinued.

10. A biomarker for diagnosing an immune-mediated inflammatory disease characterized by increased expression of MMP12, comprising MMP12, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and non-tuberculous mycobacteriosis.

11. A diagnostic agent for an immune-mediated inflammatory disease characterized by increased expression of MMP12, comprising a substance that specifically interacts with MMP12, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and non-tuberculous mycobacteriosis.

12. The diagnostic agent according to claim 11, which comprises a conjugate of a peptide having a site cleavable by MMP12 and a labeling substance.

13. The diagnostic agent of claim 11, wherein the conjugate comprises a peptide having a site cleavable by MMP12, a fluorophore attached to one end of the peptide, and a quencher attached to the other end of the peptide.

14. Human CCL-14 (hCCL-14), mouse CXCL1 (mCXCL1), mouse CXCL2 (mCXCL2), mouse CXCL3 (mCXCL3), mouse CXCL5 (mCXCL5), human CXCL1 (hCXCL1), human CXCL2 (hCXCL2), human CXCL3 (hCXCL3), human CXCL5 (hCXCL5), human CXCL6 (hCXCL6), human CXCL7 (hCXCL7), human CXCL8 (hCXCL8), human INF-α-2 (hINF-α-2), human The diagnostic agent according to claim 12, comprising a sequence of 8 to 25 consecutive amino acids, including an amino acid sequence having a site cleavable by MMP12, in a protein selected from IFN-α-6 (hIFN-α-6), human IFN-α-8 (hINF-α-8), human IFN-α-10 (hINF-α-10), human IFN-γ (hIFN-γ), and mouse IFN-γ (mIFN-γ), with the proviso that an amino acid that is cysteine (Cys) in the native sequence of the peptide may be substituted with another amino acid.

15. The diagnostic agent according to claim 12, wherein the peptide having a site cleavable by MMP12 comprises ELRCXC (X is Q or V), PLGLAG, PLGLR, FGALT, VYDLK, or PWAWR.

16. A diagnostic kit for diagnosing an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and non-tuberculous mycobacteriosis, and the kit comprises a substance that specifically interacts with MMP12 protein.

17. The diagnostic kit of claim 16, wherein the substance that specifically interacts with the MMP12 protein comprises an antibody against MMP12, an antigen-binding fragment of an antibody against MMP12, an aptamer against MMP12, or a peptide having a site cleavable by MMP12.

18. A method for screening for a substance effective in treating an immune-mediated inflammatory disease characterized by increased expression of MMP12, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitic syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and non-tuberculous mycobacteriosis, the method comprising: Measuring the level of MMP12 protein in a biological sample obtained from the subject after administration of the test substance; and If administration of the test substance reduces the protein level of MMP12, the test substance is selected as a candidate substance effective in treating an immune-mediated inflammatory disease characterized by increased expression of MMP12.

19. A therapeutic agent for an immune-mediated inflammatory disease characterized by increased expression of MMP12, comprising an MMP12 inhibitory substance, wherein the immune-mediated inflammatory disease characterized by increased expression of MMP12 is selected from the group consisting of vasculitis syndrome, granulomatous vasculitis, IgG4-related disease, inflammatory bowel disease, sarcoidosis, tuberculous mycobacteriosis, and non-tuberculous mycobacteriosis.