WO2019194217A1 - Antibody for interleukin-18 protein with high detection sensitivity and application thereof - Google Patents

Abstract

A monoclonal antibody produced by an established hybridoma expresses high selectivity for IL-18, and is also capable of performing western blotting, immunoprecipitation and immunostaining that are important for analyzing functions of IL-18. In particular, the antibody can perform western blotting with high sensitivity, and can thus detect active IL-18. In addition, the antibody inhibits the functions of IL-18, and can thus also be used for treatment.

Description

Antibody against interleukin-18 protein with high detection sensitivity and application thereof

The present invention relates to an antibody against interleukin-18 (interleukin-18, hereinafter referred to as IL-18) protein, which is an inflammatory cytokine, and its application.

IL-18 is a cytokine of the IL-1β family and is mainly expressed in macrophages, but is expressed in various cells such as dendritic cells, epithelial cells, keratinocytes. IL-18 receptor is expressed in various cells such as B cells, neutrophils, macrophages, vascular endothelial cells and smooth muscle cells in addition to NK cells, NKT cells, and CD4 T cells. The diversity of IL-18-expressing cells and receptor-expressing cells indicates the diversity of IL-18 functions and is known to be involved in various immune systems.

IL-18 has been shown to play an important role in defense mechanisms against pathogens such as parasites and bacterial infections at the time of discovery, but it also plays an important role in allergic diseases and autoimmune diseases. Is shown (Non-Patent Documents 1 and 2).

IL-18, unlike cytokines such as TNF, does not undergo production regulation at the mRNA level, and is abundant in cells as an inactive precursor (pro-IL-18). Pro-IL-18 becomes active and is released from the cell by cleaving the precursor with caspase-1 or caspase-4. Therefore, measurement of mRNA expression level and intracellular precursor protein does not measure IL-18 activity, but cleaved by caspase for functional analysis of IL-18. It is essential to analyze the activated IL-18 produced.

Since IL-18 plays an important role in various diseases, many neutralizing antibodies that bind to IL-18 have been reported (Patent Documents 1 to 10). However, since all of these documents are neutralizing antibodies developed for the purpose of treating various diseases involving IL-18, the function as neutralizing activity is disclosed.

International Publication No. 2001/058956 International Publication No. 2005/047307 International Publication No. 2007/137984 International Publication No. 2010/020593 International Publication No. 00/56771 International Publication No. 2012/085015 International Publication No. 2014/037899 JP 2000-236884 A International Publication No. 2004/097019 International Publication No. 2014/080866 International Publication No. 2012/124683

However, all of the antibodies disclosed in the above patent documents are neutralizing antibodies and do not have properties necessary for functional analysis of IL-18 such as Western blotting, immunoprecipitation, and immunostaining. . In particular, Western blotting is an important experiment in understanding the function of IL-18 because it can distinguish between IL-18 that is cleaved by caspase 1 or caspase 4 and has activity. There is no description that any of the antibodies disclosed in any of the documents can be subjected to Western blotting. Furthermore, antibodies currently marketed for research are also suitable for ELISA, and Western blotting is highly sensitive. There is nothing you can do. Although there is a report that Western blotting was performed using a commercially available polyclonal antibody and active IL-18 was detected (Non-patent Document 3), the detection sensitivity is low, and since it is a polyclonal antibody, The antibody of the same property cannot be stably obtained, and this antibody has already been discontinued. Since IL-18 is present in clinical specimens at a very low concentration, there is an antibody that can perform western blotting with high sensitivity despite the fact that detection is required with high sensitivity. Not.

An object of the present invention is to provide an antibody having high specificity for IL-18 and capable of performing Western blotting, immunoprecipitation, and immunostaining, which are important for analyzing the function of IL-18. . It is another object of the present invention to provide an antibody that inhibits the function of IL-18 and can be used not only as a research reagent but also for treatment.

The present invention includes the following monoclonal antibodies, functional fragments of monoclonal antibodies, kits containing these antibodies, genes encoding these antibodies or functional fragments, and humanized antibodies, pharmaceutical compositions, test methods, and therapeutic methods About.

(1) An anti-IL-18 monoclonal antibody that recognizes RXLFED (SEQ ID NO: 5) of human IL-18 as an epitope.
(2) The heavy chain variable domain consists of the amino acid sequence of GYAFTKYY (SEQ ID NO: 23) or GYTFTDYY (SEQ ID NO: 36), CDR1H region, INPNNGGT (SEQ ID NO: 24), or INPKNGGS (SEQ ID NO: 37). CDR2H region, CDR3H region comprising the amino acid sequence of AKLGLDFDC (SEQ ID NO: 25), light chain variable domain comprising the CDR1L region comprising the amino acid sequence of LLYSNGKTY (SEQ ID NO: 26), amino acid sequence of LVS (SEQ ID NO: 27) The anti-IL-18 monoclonal antibody according to (1), comprising a CDR3L region consisting of the CDR2L region and the amino acid sequence of VQGTHFYPYT (SEQ ID NO: 28).
(3) The amino acid sequence of the heavy chain variable region is any one of SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, and the amino acid sequence of the L chain variable region is SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. The anti-IL-18 monoclonal antibody according to (1) or (2), which is any one of them.
(4) A gene having a CDR sequence described in (2) or a gene encoding the variable region described in (3) as an open reading frame region.
(5) A functional fragment of the anti-IL-18 monoclonal antibody according to any one of (1) to (3).
(6) A kit for detecting and / or quantifying IL-18, which comprises the monoclonal antibody according to any one of (1) to (3) or the functional fragment of the monoclonal antibody according to (5).
(7) An anti-IL-18 humanized antibody comprising the CDR of (2) or the variable region of (3) and a human constant region.
(8) A pharmaceutical composition used for the treatment of an IL-18-related disease, comprising as an active ingredient the monoclonal antibody according to any one of (1) to (3) or the functional fragment of the monoclonal antibody according to (5).
(9) Detection of active IL-18 using the monoclonal antibody according to any one of (1) to (3), the functional fragment of the monoclonal antibody according to (5), or the kit according to (6) And / or a method for testing an IL-18-related disease, characterized by quantifying.
(10) A method for treating an IL-18-related disease using the pharmaceutical composition according to claim 8.

The antibodies described in this specification can detect IL-18 with higher sensitivity than currently used antibodies. In particular, since it is a highly sensitive monoclonal antibody capable of performing Western blotting, it is possible to distinguish and detect active IL-18 and precursor pro-IL-18. Moreover, since it also has IL-18 function inhibitory activity, it can also be used for treatment.

The figure which shows the specificity of monoclonal antibody 11-4.1. The figure which shows the sensitivity of the monoclonal antibody 11-4.1. The figure which shows the detection of the active form IL-18 in the adult Still disease (AOSD) patient serum by the monoclonal antibody 11-4.1. The figure which shows the detection of the active type IL-18 in the blood serum phagocytic syndrome patient serum by the monoclonal antibody 11-4.1. The figure which shows the experimental result of the immunoprecipitation by the monoclonal antibody 11-4.1. The figure which shows the experimental result of the function inhibitory activity of IL-18 by the monoclonal antibody 11-4.1. The figure which shows the experimental result of the immuno-staining by the monoclonal antibody 11-4.1. FIG. 7A is a diagram showing immunostaining by expressing activated IL-18 with a G196 tag added to HeLa cells. FIG. 7B shows the detection of endogenous IL-18 in HeLa cells by Western blotting and immunostaining. The figure which shows the result of having analyzed the recognition site | part of the monoclonal antibody 11-4.1 by Western blotting in both FIG. 8A and B. The figure which shows the result of having analyzed the detailed recognition site | part of the monoclonal antibodies 11-4.1, 4-18.1.4, and 9-4.2.1 by Western blotting. 9A shows the analysis result of 11-4.1, and FIG. 9B shows the analysis result of 4-18.1.4 and 9-4.2.1. Sensorgram of surface plasmon resonance analysis of monoclonal antibody 11-4.1.

The antibody of the present invention refers to a monoclonal antibody or derivative that specifically binds to the epitope of IL-18 shown below. It also includes functional fragments of the antibody that exhibit substantially the same antigen specificity as the original antibody. Functional fragments of antibodies include functional fragments of antibodies such as Fab, Fab ′, F (ab ′) ₂ , single chain antibodies (scFv), disulfide stabilized V region fragments (dsFv), or peptides containing CDRs. included.

In addition, humanized antibodies and genetically modified mice, in which monoclonal antibodies that specifically bind to the epitope of IL-18 identified in the present invention are transformed into human-type chimeric antibodies, humanized CDR-grafted antibodies, etc. using gene recombination technology, The human antibody used is also included in the antibody of the present invention. When administered to humans, humanized antibodies and human antibodies have fewer side effects than non-human animal antibodies, and their therapeutic effects last for a long time.

“Epitope” refers to a part of an antigen (IL-18 in the present invention) recognized by an antibody, and refers to a site on an antigen to which a domain containing an antibody variable region disclosed herein binds. means. For example, when a polypeptide such as IL-18 is used as an antigen, an antibody may recognize a linear amino acid sequence or a three-dimensional structure. And can be defined by the structure of the antigen.

As used herein, “IL-18-related disease” refers to a disease caused by excessive release of active IL-18 to the outside of a cell or a disease that may be exacerbated by IL-18. Since an IL-18-related disease is a disease that develops and exacerbates due to overexpression of IL-18, it can be treated or prevented with a drug containing an active ingredient that can inhibit the function of IL-18. it can. For example, adult Still disease (AOSD), juvenile Still disease, pancreatic cancer, lung cancer, colon cancer and other malignant tumors, cryopyrin-related periodic fever syndrome, systemic lupus erythematosus (SLE), multiple sclerosis, juvenile idiopathic Arthritis (JIA), bronchial asthma, bronchiectasis, chronic obstructive pulmonary disease (CDPD), transfusion-related 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 injury in the liver and intestine, heart failure, amyotrophic lateral sclerosis (ALS), xerophthalmia (DED) ), Keratitis, corneal neovascularization, pathological intraocular neovascularization, iritis, glaucoma, macular degeneration, Sjogren's syndrome, autoimmune uveitis, Behcet's disease, conjunctivitis, allergic conjunctivitis, eye Dermatitis, allergic rhinitis, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), steatohepatitis, ischemia-reperfusion injury, familial Mediterranean fever, TNF receptor related periodic syndrome, high IgD syndrome, gout, Schnitzler syndrome, microscopic polyangiitis, granulomatous polyangiitis, ANCA-related vasculitis consisting of eosinophilic granulomatous polyangiitis, Hashimoto's disease, Crohn's disease, ulcerative colitis, immunoglobulin-4 (IgG4) Since IL-18 has been reported to be involved in related diseases, pulmonary arterial hypertension, atopic dermatitis, etc., it is possible that therapeutic effects can be obtained with drugs that inhibit IL-18 function in these IL-18 related diseases. Be expected.

In the present specification, the term “kit” is not limited to Western blotting, immunoprecipitation, immunostaining, and IL-18 function inhibition activity test shown in the following Examples, but also includes the use of the antibody disclosed herein and IL-18. For the purpose of detecting, quantifying, or evaluating the function, a packaged combination including necessary reagents and instruments as constituent elements.

In the present invention, the test means detection of active IL-18 for the purpose of determining whether or not active IL-18 is present in a sample. Specifically, it refers to detecting whether active IL-18 is present in a sample using a patient sample. As a patient sample, samples such as plasma, serum, blood, spinal fluid, urine can be used.

Also, the pharmaceutical composition is formulated so as to be compatible with the administration route. The administration route can be formulated into a dosage form suitable for a disease such as ointment, ophthalmic preparation and the like, as well as parenteral administration such as intravenous administration, subcutaneous administration and intravitreal administration. The pharmaceutical composition can include compounds that can be generally used in the art, such as carriers, excipients, solvents, diluents, and the like suitable for the dosage form.

In the following examples, the present invention will be described in detail, but the scope of the present invention is not limited thereto.
[Example 1] Production of antibody pET28HisTEV (non-patent document) encodes a gene region encoding a polypeptide (active IL-18, SEQ ID NO: 1) at positions 37 to 193 of human inflammatory cytokine IL-18 protein (uniprot: Q14116) It was cloned into literature 4), expressed in E. coli and purified. The purified protein was immunized to mice according to a conventional method, and the purified protein used for the antigen was selected by ELISA to establish a hybridoma.

As described in detail below, three monoclonal antibodies that recognize the same epitope 11-4.1 monoclonal antibody (hereinafter referred to as 11-4.1 antibody; other antibodies are also described in the same manner), 4-18 The hybridomas 11-4.1, 4-18.1.4, and 9-4.2.1 that produce the .1.4 antibody and the 9-4.2.1 antibody were selected and analyzed. Of these monoclonal antibodies, the application of Western blotting, immunostaining, etc. will be described mainly for the 11-4.1 antibody. As shown below, the other two monoclonal antibodies have the same epitope, such as Western blotting etc. It can be used for the analysis of the same technique.

[Example 2] Evaluation of specificity of monoclonal antibody 11-4.1 produced by hybridoma 11-4.1 G196 tag (aspartic acid-leucine-valine-proline-arginine, DLVPR, hereinafter, amino acid sequence is expressed in one letter The human inflammatory cytokine IL-1β family protein fused with SEQ ID NO: 2) was expressed in human fetal kidney-derived cell line HEK-293 cells. The expressed protein was evaluated for 11-4.1 antibody using Western blotting (FIG. 1). The 11-4.1 antibody recognizes only the IL-18 protein, and the IL-1β family of IL-1α, β, Ra (Receptor antagonist), IL-33, IL-36α, β1, β2, γ, Ra IL-37 and IL-38 isoforms IL-38-1 and IL-38-2 did not react. Since it did not bind to other proteins of the IL-1β family, it can be said to be a highly specific antibody. The G196 tag is a tag sequence detected by the G196 monoclonal antibody (Patent Document 11). The Western blotting with the G196 monoclonal antibody shown in the lower part is for showing that each protein is expressed.

[Example 3] Evaluation of affinity of 11-4.1 antibody Two commercially available anti-IL-18 antibodies (D043-3: clone 25-2G, M156-3: clone 43A11, both of which are stocks) The performance was compared with the company's Medical Biology Laboratory. 100, 33, 10, 3.3 ng / mL active IL-18 protein expressed at E. coli (amino acids 37-193, indicated as cIL-18 protein in the figure) Wes (Protein Simple) And detected by capillary western immunoassay. As the primary antibody, each purified antibody was adjusted to 0.4 mg / mL and then used at the indicated dilution. That is, each primary antibody is used at a concentration of 3.2 μg / mL for 1: 125 fold dilution and 16 μg / mL for 1:25 fold dilution. All other conditions such as exposure time and secondary antibody were the same (FIG. 2).

The 11-4.1 antibody was able to detect a protein having a concentration of 3.3 ng / mL at 1:25 dilution. In contrast, at the same dilution ratio, the D043-3 antibody was able to detect a protein at a concentration of 100 ng / mL, and the M156-3 antibody was not detectable at any concentration. The 11-4.1 antibody was found to be about 60 times more sensitive than D043-3, which was confirmed to be detected.

[Example 4] Evaluation using clinical specimen (1) Evaluation using 11-4.1 antibody using clinical specimen As described above, IL-18 is produced at the mRNA level unlike cytokines such as TNF. Unregulated and abundant in cells as inactive precursors. Therefore, measurement of mRNA expression level and intracellular precursor protein does not reflect the activity of IL-18, and in order to analyze the function of IL-18, active IL-18, ie, cleaved It is essential to detect IL-18 protein. As shown in Example 3 above, since the 11-4.1 antibody is much more sensitive than the commercially available anti-IL-18 antibody, is it possible to detect active IL-18 in clinical specimens? investigated. The following research has been approved by the Ethics Committee of Shimane University School of Medicine.

IL-18 protein in the serum of adult-onset Still's disease (AOSD) patients reported to have high IL-18 protein was detected by capillary western immunoassay using Wes. As the primary antibody, the 11-4.1 antibody was adjusted to 0.4 mg / mL and then used at a 1: 125 dilution (3.2 μg / mL).

As shown in FIG. 3, the 11-4.1 antibody can detect active IL-18 in the serum of AOSD patients. Until now, there has been no report of detecting active IL-18 in patient serum by Western blotting using a monoclonal antibody, indicating that the 11-4.1 antibody is a highly sensitive and useful antibody. The bar shown below the sample number indicates that the sample is from the same patient, but not only can a small amount of active IL-18 contained in the serum be detected, but also samples 53 and 54 (FIG. 3). Top) Changes in active IL-18 before and after treatment in the same patient as in 137 and 138 (bottom of FIG. 3), and samples during and without seizures in samples 64 and 65 (upper in FIG. 3) As shown in 57 and 136 (bottom of FIG. 3), since changes in active IL-18 can be detected during a seizure and after treatment, the effect of treatment and changes in disease state can be objectively examined. That is, the 11-4.1 antibody can be used as a diagnostic agent for IL-18-related diseases. It should be noted that no patient information is obtained for the sample 52 other than that it is an AOSD case.

(2) Evaluation of 11-4.1 antibody using clinical specimens Regarding serum of patients with hemophagocytic syndrome using monoclonal antibody 11-4.1 and reported to have high levels of IL-18 protein in blood Analysis was performed.

IL-18 protein in the sera of patients with hemophagocytic syndrome was detected by capillary western immunoassay using Wes as described above. As a primary antibody, 11-4.1 was adjusted to 0.4 mg / mL and then used at a 1: 125 dilution. As an internal standard protein, active IL-18 protein cleaved with active ^caspase 4 ^105-377 was used at concentrations of 40, 20, 10, 5, and 2.5 ng / mL. The results are shown in FIG. In 2 of 6 patients (# 2, # 5), protein was detected at the same position as active IL-18, ie, active IL-18 cleaved by caspase.

ELISA can detect IL-18 with enhanced sensitivity, but since there is no antibody that can specifically recognize active IL-18, only active IL-18 cannot be directly detected by ELISA. It is clinically meaningful that active IL-18 can be detected in the serum of patients with AOSD or hemophagocytic syndrome by confirming the molecular weight by capillary western immunoassay.

[Example 5] Immunoprecipitation method with 11-4.1 antibody IL-18 ^37-193 -G196 / a vector that is expressed by fusing a G196 tag to an active IL-18 polypeptide at positions 37 to 193 The pcDNA3 vector was introduced into HEK-293 cells to express the IL-18 ^37-193- G196 protein. The usefulness of the 11-4.1 antibody in the immunoprecipitation method was evaluated in comparison with the existing G196 tag antibody (FIG. 5). 11-4.1 antibody, G196 antibody, Flag tag antibody (M2, Sigma-Aldrich) was used as a negative control antibody, immunoprecipitation was performed, and immunoprecipitation products were detected by Western blotting with G196 rabbit polyclonal antibody.

The 11-4.1 antibody immunoprecipitated IL-18 ^37-193 -G196 protein expressed in HEK-293 cells in the ^same manner as the existing G196 antibody. That is, it was revealed that the 11-4.1 antibody is a monoclonal antibody that can specifically recognize IL-18 protein and efficiently immunoprecipitate.

[Example 6] IL-18 function inhibitory activity by 11-4.1 antibody When active IL-18 protein expressed and purified in E. coli is added to acute myeloid leukemia cell line KG-1 (JCRB0065), IFN-γ is produced. It is known that If the 11-4.1 antibody is an antibody that inhibits IL-18 function, IFN-γ production by IL-18 is inhibited. A gene region encoding a polypeptide at positions 2 to 193 of the IL-18 protein (pro-IL-18, SEQ ID NO: 3) was cloned into pET28HisTEV, expressed in E. coli and purified. In addition, the gene region encoding the polypeptide at positions 105 to 377 of human caspase 4 protein (active caspase 4, SEQ ID NO: 4) was cloned into pET28HisTEV, expressed in E. coli and purified. These two proteins were mixed to purify the active IL-18 protein (SEQ ID NO: 1). Activated IL-18 purified by the above method at 2 ng / well and antibody at a concentration of 0 to 2.0 μg were added to 3 × 10 ⁴ cells / 96 well KG-1 cells, and 36 hours later, IFN in the culture supernatant -Γ was measured using an IFN-γ detection ELISA kit (Diacron).

As shown in FIG. 6, the function of IL-18 is inhibited in a concentration-dependent manner by adding the 11-4.1 antibody to the culture solution simultaneously with the active IL-18 protein. That is, the 11-4.1 antibody acts as a neutralizing antibody that inhibits the function of IL-18. IC ₅₀ is 14.2 nM, and has an activity to inhibit the function of IL-18 at a very low concentration. Therefore, the antibody of the present invention can be used as a therapeutic agent for IL-18-related diseases.

[Example 7] Immunostaining with 11-4.1 antibody IL-18 ^37-193 -G196 / pcDNA3 vector was introduced into HeLa cells to express IL-18 ^37-193 -G196 protein. After formalin fixation, immunostaining was performed using 11-4.1 antibody as a primary antibody and Alexa488-labeled anti-mouse antibody (A-11029, Thermo Fisher Scientific) as a secondary antibody according to a conventional method. As shown in FIG. 7 (A), the 11-4.1 antibody strongly recognized IL-18 ^37-193 -G196 protein transiently expressed in HeLa cells in the cytoplasm. In addition, as control, what reacted only the secondary antibody is shown.

Next, it was examined whether endogenous IL-18 protein was expressed in HeLa cells (FIG. 7B). When the endogenous IL-18 protein in HeLa cells is analyzed by Western blotting, full-length IL-18 that is not cleaved by caspase is detected as the main molecular species (FIG. 7 (B) left). Similar to the above, when immunostaining was performed with Alexa488-labeled anti-mouse antibody using 11-4.1 antibody as the primary antibody and secondary antibody, IL-18 protein was detected in the whole cell including the nucleus of HeLa cells. The That is, it has been shown that the 11-4.1 antibody can detect endogenous IL-18 protein expressed in HeLa cells by Western blotting or immunostaining. Note that the immunostained image in FIG. 7B is obtained by further black-and-white reversal of the image that has been converted to grayscale after imaging.

As shown in FIG. 7, the antibody of the present invention was analyzed by Western blotting and immunostaining using the same antibody. I can make my presence clear. Since conventional antibodies could not perform Western blotting with high sensitivity, even if the localization in the cells was clarified, it was not possible to distinguish the molecular species of full length and active type. On the other hand, since the antibody of the present invention can be applied to various methods, multifaceted analysis can be performed.

[Example 8] Epitope analysis A plasmid was prepared by pGEX-4T-2 (GE Healthcare Japan) to express active IL-18 as a GST fusion protein 15 amino acids from the N terminus. Each plasmid was expressed in E. coli, and the sequence recognized by the 11-4.1 antibody was analyzed by Western blotting using the 11-4.1 antibody. The secondary antibody was detected with ImageQuantLAS 4000 (GE Healthcare) using a peroxidase-labeled anti-mouse antibody (710-1332, Rockland). As shown in FIG. 8 (A), the 11-4.1 antibody showed strong reactivity with the 58th to 72nd amino acids.

Furthermore, which region of the sequence from positions 58 to 72 was recognized by the 11-4.1 antibody was narrowed down and a GST fusion protein was prepared and analyzed by Western blotting (FIG. 8B). ). In addition to a protein in which amino acids 58 to 72 of IL-18 are fused with GST, 62 to 72 (lane 1), 62 to 70 (lane 2), and 64 to 72 (lane 3) A plasmid was constructed with pGEX-4T-2 to express the amino acid sequence of GST as a GST fusion protein. The antibody 11-4.1 recognizes a GST fusion protein containing the amino acid sequence from positions 62 to 70, but did not recognize the amino acid sequence from positions 64 to 72 (FIG. 8 (B) right).

Therefore, the details of the amino acids recognized by the 11-4.1 antibody in the amino acid sequence from positions 62 to 70 were examined. The 11-4.1 antibody is recognized by mutating the amino acid in the region from asparagine at position 62 to threonine at position 70 (NRPLFEDMT) of IL-18 protein expressed as a GST fusion protein in E. coli to alanine. The analysis was done. Western blotting was performed using 11-4.1 antibody and quantified with ImageQuantLAS 4000.

As shown in FIG. 9 (A), the antibody 11-4.1 is a mutant in which arginine at position 63 is changed to alanine (hereinafter referred to as R63A mutant. And the mutant (D68A) in which the aspartic acid at position 68 was changed to alanine was hardly recognized. Moreover, the L65A mutant, F66A mutant, and E67A mutant showed only about half recognition of the wild type (lane left end). In addition, the 11-4.1 antibody showed the same recognition as the wild-type peptide for the N62A mutant, P64A mutant, M69A mutant, and T70A mutant.

From the above results and the already reported crystal structure of human IL-18 (Non-patent Document 5), the 11-4.1 antibody is sterically structured and has arginine (R) and 68 at position 63 exposed on the surface. It is considered that leucine-phenylalanine-glutamic acid (LFE) from the 65th position to the 67th position is recognized centering on the aspartic acid (D) at the position.

The recognition sites were also analyzed for the other two monoclonal antibodies obtained in Example 1, the 4-18.1.4 antibody and the 9-4.2.1 antibody (FIG. 9 (B)). 4-18.1.4 antibody and 9-4.2.1 antibody, like 11-4.1 antibody, hardly recognize R63A mutant and D68A mutant, and L65A mutant, F66A mutant, Only about half of the wild type E67A mutant could be recognized. In addition, the M69A mutant and the T70A mutant were recognized in the same manner as the wild type. Therefore, it is considered that the epitopes of the three types of antibodies, the 11-4.1 antibody, the 4-18.1.4 antibody, and the 9-4.2.1 antibody, are the same. That is, these three monoclonal antibodies are antibodies that recognize RXLFED (SEQ ID NO: 5) as an epitope.

In addition, the established hybridoma was confirmed for its isotype using an IsoStripe ™ mouse monoclonal antibody isotyping kit (manufactured by Sigma-Aldrich). The isotypes of the 11-4.1 antibody, the 4-18.1.4 antibody, and the 9-4.2.1 antibody were all IgG2b and κ.

The 4-18.1.4 antibody and the 9-4.2.1 antibody have the same epitope as the 11-4.1 antibody, and, like the 11-4.1 antibody, western blotting, immunoprecipitation, immunization It can be used by various methods such as dyeing and ELISA. Moreover, it has IL-18 function inhibitory activity, such as the 11-4.1 antibody, and can be applied in clinical aspects such as treatment of diseases caused by overexpression of IL-18.

[Example 9] Affinity analysis with epitope peptide Since the epitope is clarified and the affinity of the obtained monoclonal antibody is considered to be very high, the affinity was measured by surface plasmon resonance analysis. It was. A peptide (QGNLPLFEDMTDSGSGSC, SEQ ID NO: 6) having a linker sequence GSGS and a cross-linking Cys added to the C-terminus as a spacer to the 60- to 72-position sequence containing the epitope of IL-18 protein as a sensor chip CM5 by the ligand thiol coupling method (GE Healthcare, BR100012) was immobilized and analyzed.

The purified antibody 11-4.1 was measured by single-cycle using Biacore X100 (GE Healthcare) in a 5-fold, 5-fold dilution series in the range of 0.08 nM to 50 nM (FIG. 10). Analysis was performed using bivalent analysis. The actually measured values and the conformity analysis data are almost the same, and K _D : 1.25 × 10 ⁻¹¹ M, K _a : 3.3 × 10 ⁶ M ⁻¹ S ^−1, K _d : 4.1 × 10 ⁻⁵ s ⁻¹ It was. It was confirmed experimentally that the affinity was very high.

[Example 10] Analysis of antibody genes Next, gene analysis of the above three monoclonal antibodies was performed. RNA was extracted from each of the hybridomas 11-4.1, 4-18.1.4, and 9-4.2.1, and then reverse transcribed with oligo-dT primer to prepare cDNA. The sequence of the antibody gene in the hypervariable region and the amino acid sequence of the synthesized cDNA were determined by a direct sequencing method using H and L chain primer sets.

The sequences of the H chain and L chain primers used are as follows.
H chain primer VH1-1S: 5'-ggggatcc ag gts mar ctg cag sag tcw gg-3 '(SEQ ID NO: 7)
s = g + c, m = a + c, r = a + g, w = a + t
IgG2-1AS: 5'-gggaattc ctt gac cag gca tcc tag agt ca-3 '(SEQ ID NO: 8)
L chain primer VK-1S: 5'-ggggatcc gay att gtg mts acm car wct mca-3 '(SEQ ID NO: 9)
y = c + t, m = a + c, s = g + c, r = a + g, w = a + t
CK-2AS: 5'-gggaattc gaa gat gga tac agt tgg tgc-3 '(SEQ ID NO: 10)

By analysis of the H chain gene, the base sequence of the 11-4.1 antibody (SEQ ID NO: 11), the amino acid sequence (SEQ ID NO: 12), the base sequence of the 4-18.1.4 antibody (SEQ ID NO: 13), the amino acid sequence ( SEQ ID NO: 14), 9-4.2.1 The nucleotide sequence (SEQ ID NO: 15) and amino acid sequence (SEQ ID NO: 16) of the antibody were determined. Further, by analysis of the L chain gene, the base sequence of the 11-4.1 antibody (SEQ ID NO: 17), the amino acid sequence (SEQ ID NO: 18), the base sequence of the 4-18.1.4 antibody (SEQ ID NO: 19), the amino acid The sequence (SEQ ID NO: 20), the base sequence of the 9-4.2.1 antibody (SEQ ID NO: 21), and the amino acid sequence (SEQ ID NO: 22) were determined.

The amino acid sequence and gene sequence of each complementarity determining region (CDR) of these antibodies are summarized below.

[Table 1] Amino acid sequence of CDR of 11-4.1 antibody

[Table 2] Base sequence of CDR of 11-4.1 antibody

[Table 3] Amino acid sequence of CDR of 4-18.1.4 antibody

[Table 4] CDR sequence of 4-18.1.4 antibody

[Table 5] 9-4.2.1 CDR amino acid sequence of antibody

[Table 6] 9-4.2.1 CDR sequences of antibodies

As a result of gene analysis, the amino acid sequences of the CDR portions of hybridomas 11-4.1 and 4-18.1.4 were completely identical. In addition, the amino acid sequences of the heavy chain and light chain variable regions of 11-4.1 and 4-18.1.4 differed only in one place, and the other regions were identical as far as they were compared. Hybridomas 11-4.1 and 9-4.2.1 had some differences in the amino acid sequences of CDR1 and CDR2 of the heavy chain, but all other CDRs were identical.

Therefore, it was concluded that any antibody having these CDRs binds to the same epitope and exhibits similar properties. As described above, the antibody disclosed in the present specification can be used not only for various experiments such as Western blotting, immunoprecipitation, immunostaining, but also inhibit the function of IL-18. It is a very useful antibody.

Claims (9)

1. Anti-IL-18 monoclonal antibody that recognizes RXLFED (SEQ ID NO: 5) of human IL-18 as an epitope.
2. A heavy chain variable domain is a CDR1H region consisting of an amino acid sequence of GYAFTKYY (SEQ ID NO: 23) or GYTFTDYY (SEQ ID NO: 36);
   CDR2H region consisting of the amino acid sequence of INPNNGGT (SEQ ID NO: 24) or INPKNGGS (SEQ ID NO: 37),
   A CDR3H region consisting of the amino acid sequence of AKLGLDFC (SEQ ID NO: 25),
   A CDR1L region whose light chain variable domain consists of the amino acid sequence of LLYSNGKTY (SEQ ID NO: 26);
   CDR2L region consisting of the amino acid sequence of LVS (SEQ ID NO: 27),
   The anti-IL-18 monoclonal antibody according to claim 1, comprising a CDR3L region consisting of the amino acid sequence of VQGTHFYPYT (SEQ ID NO: 28).
3. The amino acid sequence of the heavy chain variable region is any one of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16,
   The anti-IL-18 monoclonal antibody according to claim 1 or 2, wherein the amino acid sequence of the light chain variable region is any one of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22.
4. A gene having the CDR sequence according to claim 2 or the gene encoding the variable region according to claim 3 as an open reading frame region.
5. A functional fragment of the anti-IL-18 monoclonal antibody according to any one of claims 1 to 3.
6. The monoclonal antibody according to any one of claims 1 to 3, or the functional fragment of the monoclonal antibody according to claim 5,
   A kit for detecting and / or quantifying IL-18.
7. An anti-IL-18 humanized antibody comprising the CDR of claim 2 or the variable region of claim 3 and a human constant region.
8. Containing the monoclonal antibody according to any one of claims 1 to 3 or the functional fragment of the monoclonal antibody according to claim 5 as an active ingredient,
   A pharmaceutical composition for use in the treatment of IL-18 related diseases.
9. Using the monoclonal antibody according to any one of claims 1 to 3, the functional fragment of the monoclonal antibody according to claim 5, or the kit according to claim 6,
   A method for testing an IL-18-related disease, which comprises detecting and / or quantifying active IL-18.

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