JP2025082445A - Active il-18 detection kit and method for running test - Google Patents

Abstract

[Problem] The objective of the present invention is to provide a simple method and kit capable of detecting only active IL-18. [Solution] A method and kit for immunologically measuring only active IL-18 using an antibody that recognizes a neoepitope generated with IL-18 activation have been constructed. An investigation was carried out using multiple antibodies that recognize the activation end of IL-18 and multiple antibodies that specifically recognize IL-18, and it was revealed that the combination of monoclonal antibody 9-10.2, which recognizes the activation end of IL-18, and a rabbit polyclonal antibody that specifically recognizes IL-18, provides the highest detection sensitivity for detecting active IL-18. The immunological measurement method and measurement kit using this combination can detect active IL-18 at low concentrations, at the pg level. [Selected Figure] Figure 7

Description

The present invention relates to a kit and a testing method that specifically recognizes active IL-18. In particular, the present invention relates to an ELISA kit that is widely used in clinical practice.

IL-18 has been suggested to be involved in various diseases. IL-18 is a cytokine of the IL-1β family, and is mainly expressed in macrophages, but is also expressed in various cells such as dendritic cells, epithelial cells, and keratinocytes. IL-18 receptors are also expressed in various cells such as NK cells, NKT cells, and CD4 T cells, as well as B cells, neutrophils, macrophages, vascular endothelial cells, and smooth muscle cells. The diversity of IL-18-expressing cells and receptor-expressing cells indicates the diversity of IL-18's functions, and it is known to be involved in various immune systems.

Due to its wide involvement in the immune system, IL-18-related diseases that develop or worsen due to overexpression of IL-18 include a variety of diseases. For example, adult-onset Still's disease (AOSD), systemic juvenile idiopathic arthritis (sJIA), malignant tumors such as pancreatic cancer, lung cancer, and colon cancer, cryopyrin-associated periodic fever syndrome, systemic lupus erythematosus (SLE), multiple sclerosis, juvenile idiopathic arthritis (JIA), bronchial asthma, bronchiectasis, chronic obstructive pulmonary disease (COPD), transfusion-associated lung injury, bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), interstitial lung disease (ILD), idiopathic pulmonary fibrosis, cystic fibrosis, rheumatoid arthritis, metabolic bone disease, severe organ damage in the liver and intestine, heart failure, amyotrophic lateral sclerosis (ALS), xerophthalmia (DED), keratitis, corneal neovascularization, pathological intraocular neovascularization, iritis, glaucoma, and macular degeneration. These include inflammatory bowel disease, Sjögren's syndrome, autoimmune uveitis, Behçet's disease, conjunctivitis, allergic conjunctivitis, eyelid dermatitis, allergic rhinitis, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), steatohepatitis, ischemia-reperfusion injury, familial Mediterranean fever, TNF receptor-associated periodic syndrome, hyper-IgD syndrome, gout, Schnitzler's syndrome, ANCA-associated vasculitis consisting of microscopic polyangiitis, granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis, Hashimoto's disease, Crohn's disease, ulcerative colitis, immunoglobulin-4 (IgG4)-associated disease, pulmonary arterial hypertension, atopic dermatitis, and NLRC4-associated autoinflammatory disease (or NLRC4 inflammasome disease).

Unlike cytokines such as TNF, IL-18 is not regulated at the mRNA level in many cells and exists abundantly within the cells as an inactive precursor (pro-IL-18). When the precursor is cleaved by caspase-1 or caspase-4, pro-IL-18 becomes active and is released outside the cell. Therefore, measuring the amount of mRNA expression or the intracellular precursor protein does not measure the activity of IL-18, and in order to analyze the function of IL-18, it is essential to analyze the active IL-18 cleaved by caspases.

As mentioned above, there are many diseases in which IL-18 is suggested to be involved, but not only active IL-18 has been measured; many cases include cases in which only precursor IL-18 has been detected, and the details of the extent of its involvement have not been clarified. Previously, attempts have been made to detect active IL-18 by distinguishing between active and precursor IL-18 based on their molecular weight using the Western blot method. However, the Western blot method is unable to detect IL-18, which is present in only about pg per ml, and even in diseases in which IL-18 is suspected to be involved, the presence of active IL-18 has not yet been tested in clinical practice.

The present inventors have prepared an antibody that recognizes only active IL-18 and disclosed its application to the treatment of inflammatory bowel disease (Patent Documents 1 and 2). In IL-18-related diseases, analyzing the increase in active IL-18 leads to prediction of the therapeutic effect of antibody drugs that can neutralize active IL-18, so a method for accurately and easily measuring active IL-18 in patient serum is required. Therefore, there has been a demand for detecting only active IL-18 by methods widely used in clinical practice, such as ELISA, using an antibody that recognizes only active IL-18. However, in reality, it has not been possible to construct a kit that can detect active IL-18 with high sensitivity.

Despite using an antibody that recognizes active IL-18, it is thought that the reason active IL-18 cannot be detected accurately is not only because the concentration of active IL-18 in the sample is low, but also because the sample contains a very large amount of precursor IL-18. Therefore, even when using an antibody that recognizes only active IL-18, the detection sensitivity is low, and it has not yet been put to practical use as a clinical test.

International Publication No. 2020/116423 International Publication No. 2023/286694 International Publication No. 2012/124683

As mentioned above, there has been no simple method for measuring only active IL-18 until now. The objective of the present invention is to provide a simple method and kit that can detect only active IL-18.

The following detection kit and detection method can be used to detect active IL-18 with high sensitivity.
(1) A test kit that specifically detects only active IL-18, which detects active IL-18 by immunological assay using a monoclonal antibody that recognizes the active end of IL-18 and an antibody that recognizes a region of active IL-18 other than the active end.
Specific detection of only active IL-18 has been required for analyzing the involvement of active IL-18 in IL-18-related diseases and for future treatment using active IL-18 antibodies. However, because the concentration of active IL-18 in serum is very low and because cells contain a large amount of precursors, no kit has been put to practical use until now. The present inventors have produced a monoclonal antibody that recognizes the active IL-18 stump, and have succeeded in developing the kit.

(2) The test kit according to (1), characterized in that the monoclonal antibody recognizing the active IL-18 stump is a 9-10.2 antibody, and the antibody recognizing a region other than the active IL-18 stump is a polyclonal antibody.
Until now, there has been no simple kit that specifically detects only active IL-18. The present inventors have investigated combinations of multiple antibodies that recognize the active stump of IL-18 and multiple antibodies that recognize regions other than the stump, and have investigated combinations that can detect active IL-18 with high sensitivity. As a result, they have revealed that active IL-18 can be detected with high sensitivity by combining the 9-10.2 antibody, which recognizes the active stump, with a polyclonal antibody, and have constructed a kit.

(3) The test kit according to (2), wherein the test kit is an ELISA kit, and when the 9-10.2 antibody is used as the solid-phase antibody, a polyclonal antibody is used as the detection antibody, and when the polyclonal antibody is used as the solid-phase antibody, the 9-10.2 antibody is used as the detection antibody.
The combination of the above two antibodies can detect active IL-18 with high sensitivity and specificity. When made into a widely used ELISA kit, the kit is also useful as a clinical test kit for testing IL-18-related diseases, etc.

(4) A method for specifically detecting only active IL-18, which is characterized by detecting active IL-18 by immunological assay using a monoclonal antibody that recognizes the active end of IL-18 and an antibody that recognizes a region of active IL-18 other than the active end.
There is no simple method for specifically detecting only active IL-18, and there has been a demand for a simple method for clinical use, such as for testing IL-18-related diseases. Furthermore, when an anti-active IL-18 antibody is used as a pharmaceutical composition in the future, it will be necessary to measure the concentration of active IL-18 in patient serum. By using a combination of an antibody that recognizes the active cleavage end and an antibody that recognizes another region, it will be possible to detect only active IL-18.

(5) The detection method according to (4), wherein the monoclonal antibody recognizing the active IL-18 truncation is 9-10.2 antibody, and the antibody recognizing a region other than the active IL-18 truncation is a polyclonal antibody.
By combining the 9-10.2 antibody, which recognizes the active stump, with a polyclonal antibody, active IL-18 can be detected sensitively and simply.

(6) The detection method according to (5), characterized in that the detection method is ELISA, and when the 9-10.2 antibody is used as the solid-phase antibody, a polyclonal antibody is used as the detection antibody, and when the polyclonal antibody is used as the solid-phase antibody, the 9-10.2 antibody is used as the detection antibody.
Since ELISA is a method that is widely used both in clinical practice and in research settings, the present method that enables detection by ELISA is a very useful method.

1 shows an example of a competition experiment using an antibody that recognizes active human IL-18, and shows the results of a competition experiment between 9-10.2 antibody and 8-4.1 antibody (top), and between -10.2 antibody and anti-G196 tag antibody. Study of antibody combinations 1. The detection antibody was fixed to the 9-10.2 antibody, and the results of studying combinations of immobilized antibodies are shown in the figure. Study of antibody combinations 2. The figure shows the results of studying combinations of detection antibodies when the solid-phase antibody was fixed to the 9-10.2 antibody. Examination of antibody combinations 3. A diagram showing the results of examination of combinations using 9-10.2 antibody and rabbit polyclonal antibody. FIG. 1 shows a calibration curve prepared using active IL-18 as a standard substance. FIG. 1 shows the results of a comparative study of serum active IL-18 concentrations in polymyositis/dermatomyositis (PM/DM) patients and healthy subjects. FIG. 1 shows the results of measuring the concentration of active IL-18 in the serum of Crohn's disease patients resistant to treatment with an anti-TNF-α antibody preparation.

Active IL-18 refers to an IL-18 molecule (SEQ ID NO: 2) in which the precursor pro-IL-18 (uniprot: Q14116, SEQ ID NO: 1) is cleaved by caspase 1/4 into peptides at positions 37 to 193. An antibody that recognizes only active IL-18 refers to an antibody that recognizes the stump sequence, i.e., the neoepitope, generated by cleavage by caspase 1/4. In this specification, an antibody that recognizes only active IL-18 is referred to as an antibody that recognizes the active stump, or an antibody that recognizes the neoepitope, or simply an activated stump antibody.

Although the specification specifically discloses the construction of an ELISA kit, any method may be used as long as it combines the two types of antibodies shown below. As the solid phase, beads may be used instead of plates, and as the detection system, antibodies labeled with radioisotopes or fluorescent dyes may be used instead of antibodies labeled with enzymes. It goes without saying that known methods used in this field, such as color development using chromogenic substrates and luminescent substances, may also be used.

As shown below, a rabbit polyclonal antibody that specifically binds to IL-18 is used in constructing the sandwich ELISA kit. However, any polyclonal antibody will do, and polyclonal antibodies obtained by immunizing animals other than rabbits, such as goats and sheep, can also be used.

[Construction of human active IL-18 ELISA kit]
1. Selection of active stump antibodies In order to construct an ELISA kit for measuring human active IL-18, selection of active stump antibodies was performed. Using several types of monoclonal antibodies that recognize the active stump of human IL-18, a competitive experiment was performed against the active stump peptide (YFGKLESKC, SEQ ID NO: 3). Here, the results of a study using 9-10.2 and 8-4.1 antibodies are shown as an example (Figure 1). The active stump peptide was crosslinked with BSA, adjusted to 5 ng/100 μl using 50 mM NaHCO ₃ , and coated on an ELISA plate overnight at 4°C. The ELISA plate was blocked and washed in the usual manner, and 9-10.2 or 8-4.1 antibody solutions adjusted to 0, 100, 300, or 1000 ng/100 μl were added, and the reaction was carried out at 25°C for 1 hour. Note that only washing solution was added to wells that did not contain antibodies.

Next, each HRP-labeled antibody (0.5 μg/μl) was diluted 500-fold with the washing solution, and 100 μl of HRP-labeled 9-10.2 antibody was added to the wells in which 8-4.1 antibody was bound to the peptide as the primary antibody, and 100 μl of HRP-labeled 8-4.1 antibody was added to the wells in which 9-10.2 antibody was bound to the peptide, and incubated at 25°C for 1 hour. After that, washing was performed, coloring was performed, and absorbance was measured (Figure 1, top). Both antibodies were shown to bind to the peptide and compete with each other, but the 9-10.2 antibody was shown to bind more strongly to the peptide. In addition, a competition experiment was performed between a monoclonal antibody (mAbProtein Co., Ltd.) against the G196 tag (DLVPR, SEQ ID NO: 4, Patent Document 3) and the 9-10.2 antibody, and an antibody against the G196 tag, which is a sequence unrelated to IL-18, was not able to competitively inhibit the binding (Figure 1, bottom).

2. Consideration of Antibody Combinations Based on the above results, it was decided to use the 9-10.2 antibody, which has a higher sensitivity as an antibody that recognizes active IL-18, as a detection antibody or a solid-phase antibody, and a consideration was given to antibodies to be combined with it.

(1) Study of combinations using 9-10.2 antibody as detection antibody First, the detection antibody was the highly sensitive 9-10.2 antibody, and the detection sensitivity was examined using the immobilized antibodies 11-4.1, 7-6.3, 7-2.5, 9-4.2, 4-18.1, and 7-12.3, which recognize the amino acid sequence at positions 58 to 72 of IL-18, and 6-2.6 and 12-7.6, which recognize the amino acid sequence at positions 128 to 142 of IL-18 (FIG. 2). The immobilized antibodies adjusted to 0.5 μg/100 μl or 1 μg/100 μl were coated on an ELISA plate overnight at 4° C. Then, the antibodies were reacted with the activated IL-18 protein adjusted to 2 ng/100 μl. After reaction with HRP-labeled 9-10.2 antibody solution (0.5 μg/μl diluted 500-fold) at 25°C for 1 hour, the enzyme substrate was added and measurement was performed in the same manner as above. The active IL-18 protein was obtained by mixing precursor IL-18 protein expressed and purified in E. coli with active caspase-4 ^105-377 also expressed and purified in E. coli. The ELISA plate was blocked by a conventional method and washed with a washing solution between each step. As shown in Figure 2, when the 9-10.2 antibody was used as the detection antibody, the highest detection sensitivity was obtained when the 7-12.3 antibody was used as the immobilized antibody.

(2) Study of combinations using 9-10.2 antibody as solid-phase antibody Next, the solid-phase antibody was fixed to the 9-10.2 antibody, and the 11-4.1, 9-4.2, 4-18.1, and 7-12.3 antibodies, which recognize the amino acid sequence at positions 58 to 72 of IL-18, and the 6-2.6 antibody, which recognizes the amino acid sequence at positions 128 to 142, were HRP-labeled and used as detection antibodies in a study similar to that described above (Figure 3). Even when the 9-10.2 antibody was used as the solid-phase antibody, the highest detection sensitivity was obtained when it was combined with the 7-12.3 antibody.

(3) Study on the combination of rabbit polyclonal antibody and 9-10.2 antibody Polyclonal antibodies are a mixture of antibodies that detect multiple epitopes, and therefore often have higher detection power than monoclonal antibodies. Therefore, the detection sensitivity was studied by combining a polyclonal antibody with 9-10.2 antibody. The polyclonal antibody used was an antibody (mAbProtein Co., Ltd.) that was obtained by immunizing a rabbit with IL-18 protein from the activation stump to the C-terminus and purifying it with Protein A. For comparison, the monoclonal antibody 7-12.3 antibody, which had the highest detection sensitivity in the above study, was used.

Measurements were performed in the same manner as above, using the 9-10.2 antibody as the detection antibody, the 7-12.3 antibody, which was the combination with the highest detection sensitivity in the above experimental results, and the IL-18 polyclonal antibody as the immobilized antibody (Figure 4). As a result, the detection sensitivity was higher when the polyclonal antibody was used as the immobilized antibody than when the 7-12.3 antibody was used. The best combination was the IL-18-specific rabbit polyclonal antibody as the immobilized antibody and the 9-10.2 antibody as the detection antibody. The amino acid sequence of the H chain (SEQ ID NO: 5) and the L chain (SEQ ID NO: 6) of the 9-10.2 antibody have already been determined and disclosed.

Activated IL-18, serially diluted from 5000 pg/ml or 1000 pg/ml, was measured using an IL-18-specific rabbit polyclonal antibody as the immobilized antibody and 9-10.2 antibody as the detection antibody. The correlation between absorbance and concentration was analyzed using the analysis software Prism (GraphPad Software) (Figure 5). Activated IL-18 was detected at concentrations of 50 pg/ml or less. By creating a calibration curve using activated IL-18 as the standard substance, the concentration of activated IL-18 in the sample can be quantified. This ELISA can also capture changes in pg concentration of activated IL-18. Therefore, it is possible to measure and analyze activated IL-18 in patient serum in clinical practice.

Next, we measured whether the ELISA kit constructed above could detect active IL-18 in patient samples.

Example 1: Measurement of active IL-18 in serum of patients with polymyositis/dermatomyositis (PM/DM) Using the ELISA kit constructed above, the serum IL-18 concentrations of 152 patients with polymyositis/dermatomyositis and 34 healthy subjects were measured (FIG. 6). The serum IL-18 concentrations of each group were compared between the two groups by Mann-Whitney U test. As a result, a significant difference in the active IL-18 concentration was observed between patients with polymyositis/dermatomyositis (IIM in FIG. 6) and healthy subjects (HC in FIG. 6) (P<0.0001). Although the results are not shown here, the active IL-18 concentration in serum of patients with polymyositis/dermatomyositis complicated with interstitial lung disease (ILD) (125 cases) tended to be higher than the active IL-18 concentration in serum of patients without ILD (27 cases), although there was no statistically significant difference.

Example 2: Measurement of active IL-18 in serum of Crohn's disease patients resistant to anti-TNF-α antibody preparations The serum of 31 Crohn's disease patients in the active stage who were resistant to anti-TNF-α antibody preparations and administered anti-IL12/23 antibody preparations (ustekinumab) was used. Patients were classified into responders (17 patients) and non-responders (14 patients) based on their therapeutic reactivity to anti-IL12/23 antibodies. Responders were defined as patients whose Clinical Disease Activity Index (CDAI) at 24 weeks after the start of treatment was reduced by 100 or more compared to the value before the start of treatment, or patients whose CDAI was less than 150. Patient background is as shown in Table 1. Before the start of treatment, non-responders had significantly lower CDAI scores and higher serum hematocrit values compared to responders.

The active IL-18 concentrations in responders and non-responders were measured using this ELISA kit (Figure 7). The concentration of active IL-18 in the serum of non-responders was significantly higher (P<0.01) than that of responders. This result suggests that treatment with anti-active IL-18 antibodies is effective for patients (non-responders) who are resistant to anti-TNF-α antibody preparations and who have not shown a therapeutic effect with anti-IL12/23 antibodies.

As shown above, active IL-18 can be detected with high sensitivity by combining rabbit polyclonal antibody and 9-10.2 antibody. Until now, there has been no simple method for detecting active IL-18, and this method will be a very useful tool for selecting patients for treatment with anti-active IL-18 antibodies in the future. In addition, there are many diseases that have traditionally been said to be IL-18-related, but there are many unknowns regarding the involvement of IL-18. It is expected that the use of this kit will also advance research into the onset of IL-18-related diseases. Furthermore, since the sequence of the IL-18 activation stump is the same in monkeys, it can also be applied to experimental monkeys.

Claims (6)

A test kit for specifically detecting only active IL-18, comprising:
A monoclonal antibody that recognizes the active end of IL-18;
A test kit that detects active IL-18 by immunoassay using an antibody that recognizes a region other than the active stump. The monoclonal antibody that recognizes the active IL-18 stump is the 9-10.2 antibody,
2. The test kit according to claim 1, wherein the antibody that recognizes a region other than the active stump of active IL-18 is a polyclonal antibody. The test kit is an ELISA kit,
9-10. When antibody 2 is used as the immobilized antibody, a polyclonal antibody is used as the detection antibody.
3. The test kit according to claim 2, wherein when a polyclonal antibody is used as the immobilized antibody, the 9-10.2 antibody is used as the detection antibody. A method for specifically detecting only active IL-18, comprising the steps of:
A monoclonal antibody that recognizes the active end of IL-18;
A detection method comprising the step of detecting active IL-18 by immunological assay using an antibody that recognizes a region other than the active stump of active IL-18. The monoclonal antibody that recognizes the active IL-18 stump is the 9-10.2 antibody,
The method according to claim 4, wherein the antibody recognizing a region other than the active cleavage end of active IL-18 is a polyclonal antibody. The detection method is ELISA,
9-10. When antibody 2 is used as the immobilized antibody, a polyclonal antibody is used as the detection antibody.
6. The method according to claim 5, wherein when a polyclonal antibody is used as the immobilized antibody, the 9-10.2 antibody is used as the detection antibody.