Autoantibodies associated with IgA nephropathy

Abstract

Provided is a therapeutic agent for diagnosing and preventing IgA nephropathy, the therapeutic agent searching for novel components that contribute to the mechanism of development of the disease.　The present invention is a method for measuring autoantibodies related to IgA nephropathy, the method being characterized in that the amount of anti-spectrin-β IgA antibodies in a sample derived from a living body is measured.

Description

[Technical Field]

[0001] The present invention relates to an autoantibody associated with IgA nephropathy and uses thereof. [Background technology]

[0002] IgA nephropathy is the most common primary glomerulonephritis in the world, and is particularly prevalent in Asian countries, including Japan. If left untreated, approximately 30-40% of cases progress to end-stage renal failure, resulting in a poor prognosis and making it a designated intractable disease (Intractable Disease Designation 66).

[0003] IgA nephropathy is defined as a chronic glomerulonephritis characterized by proliferative changes in glomerular mesangial cells and matrix, and IgA-based deposits in the mesangial region. Recently, it has been demonstrated that the IgA deposited in the glomeruli of IgA nephropathy is galactose-deficient IgA1 (Gd-IgA1), which lacks galactose in its hinge O-linked glycan. This highly disease-specific IgA1 is therefore considered to be the first hit in the development of this disease. On the other hand, it has been found that Gd-IgA1 is present in the blood of blood relatives of IgA nephropathy patients who have not yet developed nephritis and in healthy individuals, suggesting that blood Gd-IgA1 levels alone are insufficient for the onset of the disease.Furthermore, it has been argued that Gd-IgA1 may not be directly involved in the onset of this disease (Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] B Wehbi, et al. J Am Soc nephrol 2019;30:1238-1249 [Non-patent document 2] A Takahata, et al. J Am Soc nephrol 2020;31(9),2013-2024 Summary of the Invention [Problem to be solved by the invention]

[0005] As mentioned above, the components directly involved in the onset of IgA nephropathy have not yet been clarified, and there is a need to search for new components involved in the mechanism of progression of IgA nephropathy. Therefore, an object of the present invention is to search for new components involved in the mechanism of progression of IgA nephropathy and to provide a diagnostic, preventive and therapeutic drug for this disease. [Means for solving the problem]

[0006] Therefore, the present inventors hypothesized that IgA autoantibodies against glomerular components may be involved in the progression mechanism of IgA nephropathy and conducted various studies. First, the present inventors confirmed the presence of IgA autoantibodies against glomeruli in the serum of a spontaneous IgA nephropathy model mouse (gddY mouse) and human serum. Next, they isolated and analyzed proteins recognized by IgA antibodies in gddY mouse serum from glomerular proteins and identified candidate autoantigens recognized by these autoantibodies. Furthermore, they prepared recombinant proteins of these candidate proteins and examined whether they were recognized by gddY mouse serum. They found that most gddY mice had IgA autoantibodies against spectrin β, which is also present in the mesangial region, in their serum. They also confirmed that IgA autoantibodies against spectrin β were present in the serum of most IgA nephropathy patients. Furthermore, they confirmed that cytoplasmic spectrin is presented on the cell surface by MHC class II molecules. Thus, the present inventors have demonstrated that anti-spectrin βIgA antibodies are present in the serum of patients with IgA nephropathy and that these antibodies are deposited in mesangial cells in an MHC-dependent manner, thereby finding that IgA nephropathy is an autoimmune disease in that it involves tissue-specific autoantibodies. Accordingly, the present inventors have discovered that IgA nephropathy can be diagnosed by measuring the concentration of anti-spectrin βIgA antibodies in biological samples such as serum; that drugs that inhibit the activity or production of anti-spectrin βIgA antibodies are useful as preventive or therapeutic drugs for IgA nephropathy; and that screening for drugs that inhibit the activity or production of anti-spectrin βIgA antibodies allows screening for preventive or therapeutic drugs for IgA nephropathy.

[0007] That is, the present invention provides the following inventions [1] to [8]. [1] A method for measuring autoantibodies associated with IgA nephropathy, characterized by measuring the amount of anti-spectrin βIgA antibodies in a biological sample. [2] A diagnostic agent for IgA nephropathy containing an anti-spectrin βIgA antibody measurement reagent. [3] The diagnostic agent for IgA nephropathy according to [2], wherein the anti-spectrin β IgA antibody measurement reagent is an immunological measurement reagent containing spectrin β. [4] A method for screening for a preventive or therapeutic agent for IgA nephropathy, comprising screening for a drug that inhibits the activity or production of anti-spectrin βIgA antibody. [5] A preventive or therapeutic drug for IgA nephropathy, the active ingredient of which is a drug that inhibits the activity or production of anti-spectrin β IgA antibodies. [6] A drug that inhibits the activity or production of anti-spectrin βIgA antibodies, used to prevent or treat IgA nephropathy. [7] Use of a drug that inhibits the activity or production of anti-spectrin βIgA antibody for the manufacture of a drug for the prevention or treatment of IgA nephropathy. [8] A method for preventing or treating IgA nephropathy, comprising administering an effective amount of a drug that inhibits the activity or production of anti-spectrin β IgA antibody. [Effects of the Invention]

[0008] The present invention clarifies that IgA nephropathy is an autoimmune disease with tissue-specific autoantibodies, and has revealed that the autoantibodies are anti-spectrin βIgA antibodies. Furthermore, the present invention provides novel diagnostic techniques, preventive or therapeutic drugs, and screening methods for preventive or therapeutic drugs for IgA nephropathy. [Brief explanation of the drawings]

[0009] [Figure 1A] Fluorescent immunostaining of kidney sections from activation-induced cytidine deaminase (AID) knockout mice, which lack endogenous IgA antibodies, was performed using serum from 16W Balb / c (left) or gddY (right) mice as the primary antibody, and PE-labeled anti-IgA antibody was used as the secondary antibody. The green circle indicates the glomerular region. [Figure 1B] Immunoblot analysis of glomerular lysates from gddY mice with serum IgA from 12W Balb / c or 8W gddY mice (N=3) is shown. Each lane represents the serum from each mouse. [Figure 1C] Immunoblotting of mouse primary mesangial cell (MC) lysates with serum IgA from 12W Balb / c (left) or 8W gddY (right) mice (N=3) is shown. Each lane represents a serum from each mouse. [Figure 1D] 1 shows the reactivity of serum IgA from 8W-16W Balb / c or 8-16W gddY mice to p250+. [Figure 2A] Immunoblot analysis of embryonic kidney (HEK) 293T cells transfected with a mock vector (M) or a FLAG-tagged sptbn1A expression vector (1A) is shown. Serum from Balb / c (N=4) or gddY (N=4) mice was used as the primary antibody, and anti-IgA antibody was used as the secondary antibody. Overexpression of FLAG-tagged sptbn1A was confirmed with an anti-FLAG antibody (lower panel). [Figure 2B]Immunoblot analysis of HEK293T cells infected with a mock vector (M) or a FLAG-tagged sptbn1B expression vector (1B) is shown. Serum from Balb / c (N=4) or gddY (N=4) mice was used as the primary antibody, and anti-IgA antibody was used as the secondary antibody. Overexpression of FLAG-tagged sptbn1B was confirmed with an anti-FLAG antibody (lower panel). [Figure 2C] Immunoblot analysis of HEK293T cells infected with a mock vector (M) or a FLAG-tagged sptbn1C expression vector (1C) is shown. Serum from Balb / c (N=4) or gddY (N=4) mice was used as the primary antibody, and anti-IgA antibody was used as the secondary antibody. The red arrow indicates the presence of anti-Sptbn1C IgA antibody in serum from gddY mice. Overexpression of FLAG-tagged sptbn1C was confirmed using an anti-FLAG antibody (lower panel). [Figure 2D] Shown is the reactivity of serum IgA from 12-16W Balb / c (left) and 8-16W gddY (right) mice to sptbn1C. [Figure 3A] The results of immunoblotting of lysates of primary human MCs with serum from healthy individuals (healthy controls: HC) or patients with IgA nephropathy (IgAN) are shown. The red arrow indicates the presence of IgA autoantibodies against p250+ in the serum of patients with IgA nephropathy. Numbers #1 to #5 indicate the ID number of each individual. [Figure 3B] The reactivity of serum IgA from HC or IgA nephropathy patients to p250+ is shown. [Figure 3C] Immunoblot analysis of HEK293T cells infected with a mock vector (M) or a FLAG-tagged full-length SPTBN1 expression vector (S) is shown. Sera from HCs, patients with other renal diseases (disease control: DC), or patients with IgA nephropathy (IgAN) were used as the primary antibody, and anti-IgA antibody was used as the secondary antibody (upper panel). The red arrow indicates the presence of anti-SPTBN1 IgA1 antibodies in the serum of patients with IgA nephropathy. Overexpression of FLAG-tagged SPTBN1 was confirmed with an anti-FLAG antibody (lower panel). [Figure 3D] This shows the reactivity of serum IgA1 to SPTBN1 in sera from healthy individuals, patients with other nephritis, and patients with IgA nephropathy. [Figure 4A] The results of flow cytometry analysis of CD138 and IgA expression in mononuclear cells isolated from mouse kidneys (intracellular staining) are shown. The numbers adjacent to the outlined areas indicate the percentage of IgA+ plasma cells (PCs) among live cells. gddY (8-week-old), Balb / c (8-week-old), NZB / w F1 (24-week-old), and Faslpr / lpr (24-week-old) mice were used. [Figure 4B] Flow cytometry was performed to analyze Ki67 expression in the following cell populations: IgA+PCs infiltrating gddY kidneys (blue), IgA+PCs in the small intestine of C57BL / 6 mice (red), germinal center B cells in the spleen of C57BL / 6 mice immunized with the hapten 4-hydroxy-3-nitrophenylacetyl (NP)-chicken gamma globulin and alum 6 days prior to analysis (green), and naive B cells in the spleen of C57BL / 6 mice (gray). [Figure 4C] The frequencies of IgA+ PCs among live cells in the kidneys of Balb / c (blue), NZB / w F1 (green), Faslpr / lpr (purple), and gddY (red) mice are shown. Quantitation results for Figure 4A. Each point represents one mouse. In Figure 4C, **p<0.01, ***p<0.001, and ****p<0.0001 (one-way analysis of variance (ANOVA) with multiple comparisons). [Figure 4D] Figure 4C shows the number of infiltrating IgA+ PC cells per liver for the mice in Figure 4C. Each point represents one mouse. In Figure 4D, **p<0.01, ***p<0.001, ****p<0.0001 (one-way analysis of variance (ANOVA) with multiple comparison test). [Figure 4E] The frequency of IgA+ PCs in live cells from gddY mice of each age is shown. Each dot represents one mouse. [Figure 4F] Fluorescent immunostaining of gddY mouse kidneys was performed using IgA (red) and CD138 (blue). [Figure 4G]The results of analyzing the frequency of IgG+ (blue) or IgA+ (red) cells among PCs infiltrating the kidneys of gddY mice are shown in Figure 4G. ****p<0.0001 (Student's t-test). [Figure 5A] Immunohistochemical staining of a kidney biopsy section from a patient with IgA nephropathy is shown. Brown arrows indicate IgA+ cells. [Figure 5B] Serum creatinine (S-creatinine) values ​​for each patient are plotted against the number of plasma cells per glomerulus in each biopsy from the corresponding patient. [Figure 5C] The proteinuria level for each patient is plotted against the number of plasma cells per glomerulus in each biopsy for the corresponding patient. [Figure 6A] This shows a method for culturing IgA+ plasma blast cells (PBs) that infiltrate the kidneys of gddY mice. [Figure 6B] This image shows immunofluorescence staining of kidney sections from AID knockout mice. Serum IgA from 12W Balb / c mice or culture supernatant of IgA+PBs infiltrating the kidney of gddY mice was used as the primary antibody, and PE-labeled anti-IgA antibody was used as the secondary antibody. The green circle indicates the glomerular region. [Figure 6C] The results of immunoblotting of glomerular lysates using culture medium alone (Control) or culture supernatant of IgA+PBs from gddY mouse kidney (kidney) as the primary antibody and anti-IgA antibody as the secondary antibody are shown. [Figure 6D] Immunoblot analysis using HEK293T cells infected with a mock vector (M) or a FLAG-tagged sptbn1C expression vector (S) is shown. Culture supernatants from IgA+ PCs present in the lamina propria of the small intestine (SI) or IgA+ PCs infiltrating the kidney (kidney) in gddY mice were used as the primary antibody, and anti-IgA antibody was used as the secondary antibody (top panel). The red arrow indicates the presence of anti-Sptbn1C IgA antibody in the culture supernatant from IgA+ PBs infiltrating the kidney in gddY mice. Overexpression of FLAG-tagged sptbn1C was confirmed using an anti-FLAG antibody (bottom panel). [Figure 6E]Recombinant IGA antibodies were prepared from IgA+PBs infiltrating the kidneys of gddY mice. The binding of each antibody to mouse MCs was analyzed by flow cytometry (intracellular staining). As a control, an IgA antibody with affinity for NP was used (#NP). The binding of clones #5 (negative), #9 (weakly positive), and #11 (positive) to MCs was analyzed. MCs stained with only the secondary antibody (anti-IgA) are shown in gray. [Figure 6F] Binding of recombinant IgA antibodies to MCs by flow cytometry. [Figure 6G] The binding of #NP or clone #9 to sptbn1C was analyzed by Western blot (WB). HEK293T cells infected with mock vector (M) or FLAG-tagged sptbn1C expression vector (S) were immunoprecipitated with anti-FLAG antibody and immunoblotted with recombinant antibody (top panel). #NP or clone #9 was used as the primary antibody, and anti-IgA antibody was used as the secondary antibody. The red arrow indicates that clone #9 recognizes sptbn1C. Expression of FLAG-tagged sptbn1C was confirmed with anti-FLAG antibody (bottom panel). [Figure 6H] This shows the binding of a recombinant antibody prepared from IgA+PBs derived from the kidneys of gddY mice to sptbn1C. [Figure 7A] The binding of #NP or clone #9 to MCs cultured in the presence or absence of IFN-γ was analyzed by flow cytometry. MCs were cultured for 48 hours in the presence or absence of IFN-γ (50 ng / mL), and cell surface staining was performed using anti-IA (H2-S) antibody and recombinant antibody as primary antibodies, and anti-IgA antibody as secondary antibody. The number of adjacent outlined areas indicates the frequency of IA- / IgA-, IA- / IgA+, I-A+ / IgA-, or I-A+ / IgA+ cells among live cells. The histogram shows the binding of clone #9 to IA-non-expressing MCs (gray) and IA-expressing MCs (red). [Figure 7B]Human HEK293T cells expressing C57BL / 6- or gddY-derived IA were infected with a T7-tagged sptbn1C expression vector (C57BL / 6 IA HEK, gddY IA HEK). The surface expression of T7 in each cell line was analyzed by flow cytometry. [Figure 7C] C57BL / 6 IA HEK or gddY IA HEK were infected with a mock vector (M) or a T7-tagged sptbn1C expression vector (S). The I-Aβ chain (the β chain is FLAG-tagged) was immunoprecipitated from the lysate of infected cells using an anti-FLAG antibody, and the coprecipitated protein was immunoblotted with an anti-T7 antibody (top). The whole cell lysate (WCL) was immunoblotted with an anti-T7 antibody (middle), and the I-Aβ chain in the immunoprecipitated protein was immunoblotted with an anti-FLAG antibody (bottom). [Figure 8] Figure 4B shows the gating diagram for flow cytometry. [Figure 9] This figure shows gating of IgA+ PCs (CD138+B220low) infiltrating the kidneys of gddY mice. The frequency of IgA+ or IgG+ cells among plasma cells infiltrating the kidneys of gddY (upper panel) or NZB / w F1 mice (lower panel) was analyzed by flow cytometry. [Figure 10] This shows the number of somatic hypermutations in the heavy and light chain variable regions of recombinant antibodies prepared from IgA PBs infiltrating the kidneys of gddY mice. [Figure 11] Surface expression of IA on mouse MCs cultured in the presence of IFN-γ was analyzed by flow cytometry. Primary MCs were cultured in the presence or absence of IFN-γ, and after 48 hours, surface staining was performed with PE-conjugated anti-IA antibody or an isotype control antibody. [Figure 12] Expression of IA on the surface of C57BL / 6 IA HEK and gddY IA HEK was confirmed by flow cytometry. HEK 293T cells (control HEK) that do not express IA are shown in gray. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention is characterized in that the inventors have clarified that IgA nephropathy is an autoimmune disease with tissue-specific autoantibodies and have discovered that the autoantibodies are anti-spectrin β IgA antibodies. Spectrin is a major component of the protein meshwork that covers the surface of the plasma membrane of cells such as vertebrate red blood cells, and is a high-molecular-weight heterodimer consisting of two types of subunits, α and β. Spectrin is also present on the inner surface of many types of cell membranes, where it acts as a scaffold to maintain the shape of the cell and plays an important role in maintaining the structure of the cell membrane. However, it was completely unknown that anti-spectrin β IgA antibodies could be autoantigens for autoantibodies, let alone that they are autoantibodies involved in IgA nephropathy. There are five types of spectrin β (I to V), but in the present invention, an antibody that recognizes spectrin β-II (β-II spectrin, Spectrin β, Non-Erythrocytic 1), i.e., an anti-spectrin β-II IgA antibody, is preferred.

[0011] The method of the present invention for measuring an autoantibody associated with IgA nephropathy is characterized by measuring the amount of anti-spectrin βIgA antibody in a biological sample.

[0012] Examples of biological samples to be measured include serum, plasma, kidney biopsy samples, lymphatic fluid, etc., with serum, plasma, and kidney biopsy samples being preferred, and serum being particularly preferred.

[0013] The living body to be measured includes animals including humans, preferably humans. The living body may be a healthy living body or a living body suspected of having IgA nephropathy, preferably a human suspected of having IgA nephropathy.

[0014] A preferred means for measuring the amount of anti-spectrin β IgA antibody is an immunological measurement method using a reagent containing spectrin β, an autoantigen. The immunological assay may be any known immunological assay, such as radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), indirect fluorescence assay, luminescent immunoassay, physicochemical assay (TIA, LAPIA, PCIA), or Western blotting, with ELISA being preferred. The ELISA method involves reacting an antigen immobilized on a solid phase with an antibody, then reacting the antibody bound to the antigen with a secondary antibody labeled with an enzyme such as peroxidase or alkaline phosphatase, and then measuring the enzyme label by an appropriate method, such as a competitive method or a sandwich method, with the sandwich method (solid-phase sandwich method) being a particularly suitable example.

[0015] The solid-phase sandwich method is carried out, for example, as follows: First, the antigen spectrin β is solidified, and then a specimen as a test sample is added to this. As a result, an antigen-antibody reaction occurs between the solid-phase antigen and the antibody in the sample, and anti-spectrin β IgA antibodies present in the sample bind to the solid-phase antigen. Next, this bound antibody is detected using an antibody detection reagent to measure the amount of anti-spectrin β IgA antibodies present in the test sample.

[0016] In the above, the antibody detection reagent can also be solid-phased, used to capture the antibody in the specimen, and then the antigen spectrin β is added to bind to the anti-spectrin β IgA antibody in the captured antibody, and then a labeled form of the specific antibody of the antigen can be bound, thereby detecting and measuring the target anti-spectrin β IgA antibody present in the test sample.

[0017] The selection of various means for these measurement techniques and their modifications are well known to those skilled in the art, and there are no particular limitations in the present invention, and any of these techniques can be adopted (see, for example, "Outline of Clinical Testing Methods," Kanehara Publishing, 1995).

[0018] For example, a wide variety of commonly used insoluble, inert supports can be used as the solid phase in the solid-phase method, including sticks, beads, microplates, test tubes, and the like made of various materials such as glass, cellulose powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethylcellulose, ion-exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid copolymer, amino acid copolymer, and ethylene-maleic acid copolymer.

[0019] The method for immobilizing antigens or antibodies is not particularly limited, and either physical or chemical bonding can be used. Representative examples include the diazo method as a covalent bonding method, the peptide method (such as the acid amide derivative method, carboxyl chloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide-activated polysaccharide method, cellulose carbonate derivative method, and methods using condensation reagents), the alkylation method, carrier binding methods using crosslinking reagents (such as glutaraldehyde and hexamethylene isocyanate as crosslinking reagents), and methods using chemical reactions such as carrier binding methods using the Ugi reaction; ionic bonding methods using carriers such as ion exchange resins; and physical adsorption methods using porous glass such as glass beads as a carrier.

[0020] The labeling agent used in each measurement system is not particularly limited, and any known labeling agent can be used. Specific examples include various radioisotopes commonly used in immunoassays, enzymes such as alkaline phosphatase (ALP) and peroxidase (POX), fluorescent substances such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (RITC), and 1N-(2,2,6,6-tetramethyl-1-oxyl-4-piperidyl)-5N-(aspartate)-2,4-dinitrobenzene (TOPA).

[0021] In addition to the above-mentioned enzyme-labeling substances, examples of enzyme-labeling substances include microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-nase, P-nase, etc. Labeling with these labeling substances can be carried out according to known methods (see, for example, "Monoclonal Antibodies," by Iwasaki Tatsuo et al., Kodansha Scientific, 1984; "Enzyme Immunoassay," 2nd ed., by Ishikawa Eiji et al., Igaku-Shoin, 1982, etc.).

[0022] Enzyme activity can also be measured according to known methods depending on the type of enzyme used. For example, when peroxidase is used as the labeling enzyme, ABTSJ (2,2'-azino-bi(3'-ethylbenzthiazoline sulfonic acid)) is used as the substrate, and when alkaline phosphatase is used, p-nitrophenyl phosphate is used as the substrate, and the decomposition of each substrate can be measured using a spectrophotometer or the like (see, for example, "Enzyme Immunoassay," 2nd ed., Ishikawa Eiji et al., Igaku Shoin, 1982).

[0023] When a label with a radioisotope, a fluorescent substance, or the like is used instead of the enzyme label, the label can be measured by a known method.

[0024] The solvent used in the above measurement system may be any commonly used solvent as long as it does not adversely affect the reaction. Specifically, buffers with a pH of about 5-9, such as citrate buffer, phosphate buffer, Tris buffer, and acetate buffer, can be suitably used.

[0025] The conditions for the immune reaction (binding) are not particularly limited, and the usual conditions used in this type of assay can be used. The reaction can generally be carried out at a temperature of 45°C or less, preferably about 4-40°C, for about 1-40 hours.

[0026] Furthermore, when the measurement target is a kidney biopsy sample, an immunostaining method can also be employed.

[0027] If the amount of anti-spectrin βIgA antibody thus obtained is higher than the amount of anti-spectrin βIgA antibody in healthy individuals or patients with renal diseases other than IgA nephropathy, it can be determined that the biological sample is related to IgA nephropathy.

[0028] A reagent for measuring anti-spectrin β IgA antibodies, typified by the immunological assay reagent containing the above-mentioned spectrin β, is useful as a diagnostic agent for IgA nephropathy. The reagent for measuring anti-spectrin β IgA antibody is preferably an immunological assay reagent containing spectrin β, and more preferably an immunological assay reagent containing spectrin β-II.

[0029] Since anti-spectrin βIgA antibodies are autoantibodies involved in IgA nephropathy, it is clear that drugs that inhibit the activity or production of anti-spectrin βIgA antibodies are useful as preventive or therapeutic agents for IgA nephropathy, and by screening for drugs that inhibit the activity or production of anti-spectrin βIgA antibodies, it is possible to screen for preventive or therapeutic agents for IgA nephropathy.

[0030] A screening method for drugs that inhibit the activity or production of anti-spectrin β IgA antibodies includes, for example, a method in which spectrin β is reacted with anti-spectrin β IgA antibodies in the presence of a test substance, and it is confirmed that the reaction between the antigen and the antibody is inhibited by the presence of the test substance. The reaction between spectrin β and anti-spectrin β IgA antibodies can be carried out using a reaction system similar to that used in the above-mentioned immunoassay method. Alternatively, screening can be performed by culturing cells that produce anti-spectrin βIgA antibodies in the presence of a test substance and examining the effect of adding the test substance on the production of anti-spectrin βIgA antibodies.

[0031] Drugs that inhibit the activity or production of anti-spectrin βIgA antibodies selected by such screening are useful as drugs for preventing or treating IgA nephropathy. Specific examples of drugs that inhibit the activity or production of anti-spectrin βIgA antibodies include chimeric antibodies that combine the variable region of anti-spectrin βIgA antibodies with the Fab region of human IgG. The preventive or therapeutic agent for IgA nephropathy may contain a drug that inhibits the activity or production of anti-spectrin βIgA antibody, and may be in the form of a pharmaceutical composition that further contains a pharmaceutically acceptable carrier.

[0032] The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered orally, it can be formulated into dosage forms such as tablets, pills, sugar-coated tablets, soft capsules, hard capsules, solutions, suspensions, emulsions, gels, syrups, and slurries together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. When used as a parenteral administration composition, it can be formulated into dosage forms such as an injectable solution, suspension, emulsion, cream, ointment, inhalant, suppository, etc. together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. When used as an injectable composition, it can be dissolved in an aqueous solution, preferably a physiologically compatible buffer solution such as Hanks' solution, Ringer's solution, or physiological saline buffer. [Example]

[0033] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0034] Example 1 Materials and Methods (1) Mouse The gddY mice were established by selective breeding of early-onset ddY mice over 20 generations. lpr / lpr Mice were purchased from Sankyo Labo Service Co., Ltd. In this study, female NZB / W F1 and Fas lpr / lpr was used.

[0035] (2) Human subjects With informed consent and approval from the Research Ethics Committee of Juntendo University Hospital, sera from patients and volunteers with IgA nephropathy or other renal diseases, and kidney biopsy specimens from patients with IgA nephropathy were obtained at Juntendo University Hospital.

[0036] (3) Immunofluorescence and electron microscopy Isolated mouse kidneys were fixed in 4% paraformaldehyde for 6 hours and then transferred to 30% sucrose overnight at 4°C. The specimens were embedded in OCT compound (Sakura Finetek), frozen in liquid nitrogen, and subjected to fluorescent immunostaining. 6 μm-thick frozen sections were incubated with 3% BSA / PBS for 60 minutes at room temperature to block nonspecific staining. Kidney sections were stained overnight at 4°C with 1:10 diluted mouse serum as the primary antibody, followed by anti-IgA antibody for 1 hour as the secondary antibody. After washing, slides were mounted with ProLong Gold antifade reagent containing DAPI (Invitrogen). PE-goat anti-mouse IgA antibody (Abcam) was used as the anti-IgA antibody. Biotinylated anti-CD138 (Biolegend) and APC-conjugated streptavidin (Biolegend) were used to stain PCs infiltrating the mouse kidney. All samples were photographed with a confocal microscope (FV3000; Olympus). Renal biopsy specimens from patients were fixed in 15% formaldehyde, embedded in paraffin, and then 3-μm-thick sections were prepared. Immunohistochemical staining was performed after antigen retrieval using either 40 minutes of heat induction (for CD138 staining) or 0.05% bacterial protease subtilisin A (Sigma-Aldrich; for IgA staining) at room temperature for 2 hours. Slides were incubated with primary antibodies for 30 minutes (anti-IgA, DAKO Sharp IR51061; anti-CD138, 1:50, DAKO #7228) and then developed with EnVision+ System HRP-conjugated polymer anti-mouse (DAKO) for 30 minutes. The staining was then developed using the Liquid DAB+ Substrate Chromogen System (DAKO).

[0037] (4)WB method Balb / c mouse-derived MCs and human primary MCs were used to detect IgA autoantibodies in serum from gddY mice or IgAN patients, respectively. HEK293T cells were transfected with vectors expressing full-length or split mouse Sptbn1 and full-length human SPTBN1, and the resulting cell lysates were used to detect anti-spectrin β IgA antibodies in serum. WB was performed as previously described. Briefly, cells were lysed in 1% NP-40 lysis buffer containing protease inhibitors. The cell lysates were reduced and denatured with SDS, boiled, and subjected to SDS-PAGE. Subsequently, immunoblotting was performed using serum and antibodies. Mouse and human sera were used as primary antibodies at dilutions of 1:40 and 1:50, respectively. Peroxidase-conjugated anti-mouse IgA antibody, anti-human IgA antibody, or IgA1 antibody was used as detection antibodies.

[0038] (5) Immunoprecipitation To identify the autoantigens recognized by serum IgA from gddY mice, whole kidneys or isolated glomeruli from C57BL / 6 mice were lysed in 1% NP-40 lysis buffer containing protease inhibitors. Balb / c or gddY mouse serum (mixed serum from 10 mice) was reacted with a goat polyclonal anti-mouse IgA antibody coupled to NHS-activated Sepharose 4 Fast Flow (GE Healthcare) for 6 hours. Serum IgA was eluted with 0.1 M glycine-HCl pH 2.0, and the lysate was dialyzed against PBS. The purified serum IgA was then directly coupled to Pierce NHS-Activated Magnetic Beads (Thermo Fisher Scientific), which were used to immunoprecipitate glomerular proteins recognized by serum IgA.

[0039] (6)Mass spectrometry Glomerular proteins immunoprecipitated with serum IgA from Balb / c and gddY mice were separated by SDS-PAGE. The gel section containing proteins precipitated exclusively by serum IgA from gddY mice was excised and destained. The gel pieces were dehydrated in 100% acetonitrile (ACN) at room temperature for 10 minutes and then dried under vacuum. The gel pieces were then simultaneously reduced and alkylated with 10 mM Tris(2-carboxyethyl)phosphine hydrochloride (ThermoFisher) and 40 mM chloroacetamide (Sigma) in 100 mM Tris-HCl (pH 8.5) and incubated at 70°C for 5 minutes. After reduction and alkylation, the solution was removed, and the gel pieces were washed with 50% ACN for 10 minutes each while shaking at room temperature on an Intelli-Mixer RM-2M (ELMI Ltd, Latvia). The gel pieces were then dehydrated in 100% ACN for 10 minutes and dried. 20 ng / L trypsin / Lys-C (Promega) in 100 mM HEPES (pH 8.5) was added to each gel piece and kept on ice for 30 min. The gel pieces were then incubated overnight at 37 °C. After digestion, peptides were extracted from the gel pieces three times with 0.1% formic acid in 50% ACN, and the solution was collected into the same vial each time. The resulting peptides were desalted using an SDB tip (GL Science, Tokyo) and lyophilized using a centrifugal evaporator EZ-2 Elite (Genevac Ltd, Ispswich, UK). Proteins were identified by nano-LC-MS / MS using a Triple TOF 5600+ system and an Eksigent nano-LC system (AB SCIEX, MA) operated with Analyst TF 1.7 software. The obtained MS data were searched with ProteinPilot 5.0.1 software (AB SCIEX) using the UniProt database (2020_04). A 1% false discovery rate confidence cutoff was applied for protein identification.

[0040] (7) Plasmid constructs To construct expression vectors for mouse full-length or split Sptbn1, cDNA was prepared from isolated glomeruli of C57BL / 6 mice. The full-length sptbn1 sequence was amplified from these cDNAs using KOD+Neo DNA polymerase (Toyobo) and subcloned into the pCAT7 neo vector (a vector expressing a double T7-tagged protein at the N-terminus of the target protein). For mouse split sptbn1, sptbn1A (CDS 4..2364>), sptbn1B (CDS 2365..4728>), and sptbn1C (CDS 4729.7092) sequences were amplified using KOD Fx Neo or Plus Neo DNA polymerase (Toyobo) and subcloned into the pCAT7 neo vector or p3XFLAG-CM-10 (Sigma-Aldrich). The FLAG-tagged human full-length SPTBN1 expression vector was a kind gift from Dr. Imamichi (J Exp Med. 2013; 210(3): 517-34). For HA-tagged I-Aα and FLAG-tagged I-Aβ derived from gddY or C57BL / 6 mice, cDNA was prepared from isolated naive B cells of gddY or C57BL / 6 mice. The I-Aα and I-Aβ sequences were amplified from these cDNAs using KOD+Neo DNA polymerase (Toyobo) and subcloned into pMX vectors. The HA or FLAG tag sequence and a linker peptide sequence (aggtggaggcagc) were added to the N-terminus of I-Aα or I-Aβ.

[0041] (8) ELISA assay Serum total IgA levels were analyzed by sandwich ELISA according to the method described in Kidney Int. 2007;72(3):319-27.

[0042] (9) Tissue preparation, flow cytometry, and single B cell sorting Perfused kidneys were cut into 2-3 mm pieces and pulverized with 0.6 mg / mL collagenase D (Roche) and 100 μg / mL DNase I (Roche). Renal mononuclear cells were isolated by gradient centrifugation using Percoll. Mononuclear cells from the small intestinal lamina propria of gddY mice were isolated as described in Nature Communications 2019;10(1):3650. Single-cell suspensions were stained with the following reagents: BD Biosciences: B220 (APC-Cy7), CD138 (PE or BV421), GL7 (PerCP Cy5.5), Ki67 (PE-Cy7), IgG1, G2a, G2b, G2c, and G3 (FITC), IgA (biotin), and streptavidin (VB421 or APC). Intracellular staining of IgA PBs and IgG PCs was performed using a Fixation and Permeabilization solution kit (BD Biosciences), and intracellular staining of Ki67 was performed using a Foxp3 Staining Buffer Set (eBioscience). To isolate single IgA PBs from the kidneys of gddY mice, mononuclear cells isolated from the kidneys were surface stained with CD138 (PE), IgA (biotin), and streptavidin (APC). Single CD138+IgA+ PBs were isolated individually in a 96-well PCR plate (Eppendorf) containing 4 μL of lysis solution. Surface expression of IA on MCs was confirmed using an anti-IA antibody (PE, Southern Biotech). All samples were analyzed using a FACS Calibur, FACS Aria II, or FACS Canto II (BD Biosciences). Data were analyzed using FlowJo (Tree Star).

[0043] (10)Cell culture IgA+ PBs derived from the kidney or small intestine of gddY mice were cocultured with feeder cells expressing CD40L, B-cell activating factor, and aproliferation-inducing ligand in B cell medium (RPMI-1640 medium (Wako)) supplemented with interleukin-6 (IL-6), 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 5.5 × 10-5 M 2-ME, 100 U / mL penicillin, and 100 μg / mL streptomycin (GIBCO) in a 5% CO2, 37°C incubator. The culture supernatants of IgA+ PBs were collected on day 6. MC cells isolated from Balb / c mice were used. MCs were cultured in Dulbecco's modified Eagle's medium (High Glucose) containing L-glutamine, phenol red, and sodium pyruvate (DMEM) supplemented with 10% FBS, 10 mM HEPES, 100 U / mL penicillin, and 100 μg / mL streptomycin. MCs were used between passages 8 and 16.

[0044] (11) ADDIN EN.CITE ADDIN EN.CITE.DATA Generation of recombinant antibodies Recombinant antibodies from IgA PBs in gddY mouse kidneys were produced as described in J Immunol Methods. 2009;350(1-2):183-93. Using the superPrep II Cell Lysis & RT Kit (TOYOBO), total RNA extracted from isolated IgA+ PBs from gddY mouse kidneys was reverse-transcribed (RT) to synthesize cDNA in a 96-well plate (final volume 20 μL / well) containing nuclease-free water (Eppendorf). RT was performed at 42°C for 5 minutes, 25°C for 10 minutes, 50°C for 60 minutes, and 94°C for 5 minutes. The cDNA was stored at -20°C until use. The IgA heavy chain gene and Igκ or Igλ light chain gene were amplified by nested PCR (40 cycles of 98°C for 10 seconds, 55°C for 30 seconds, and 68°C for 30 seconds). After identification of IgV and J genes using IgBlast (http: / / www.ncbi.nlm.nih.gov / igblast / ) and VBASE2 (http: / / www.vbase2.org / ), a second PCR reaction was performed using 0.5 μL of the unpurified first-round PCR product as a template and single-gene-specific V and J gene primers containing restriction sites (variable region PCR product). The number of somatic mutations in the V genes was counted, including FWR1-FWR3. The variable region PCR product and the pCAGGS expression vector containing the mouse IgA, Igκ, or Igλ constant region were digested with restriction enzymes and then ligated using Ligation High Ver. 2 (TOYOBO). This plasmid was transformed into competent E. coli HST08 bacteria via heat shock at 42°C and cultured on ampicillin plates (100 μg / mL). After purifying the expression vector from the colony, the PCR product was sequenced to confirm its identity with the second-round PCR product. HEK293T cells were transiently transfected with equal amounts of 1 μg each of the heavy and light chain expression vectors. Three days after transfection, the supernatant was collected and replaced with fresh medium. Six days after transfection, the culture supernatant was collected again. The culture supernatant was removed from cell debris by centrifugation at 1400 rpm for 10 minutes and stored at -20°C until use.As a negative control, a recombinant IgA antibody against NP was prepared, which had VH 186.2, DFL 16.1, JH 2 of the heavy chain, and λ1 of the light chain.

[0045] (result) (1) The serum of gddY mice contains IgA-type autoantibodies against mesangial cells. Antibody deposition in different tissues suggests antigen recognition by autoantibodies. Therefore, we first examined the possibility of the presence of IgA autoantibodies against glomerular MCs in gddY mouse serum. Kidney sections from AID-deficient mice lacking endogenous IgA antibodies were immunofluorescently stained using Balb / c or gddY mouse serum as the primary antibody and anti-mouse IgA as the secondary antibody. Only serum IgA from gddY mice, but not from Balb / c mice, bound to glomeruli (Figure 1A), indicating the presence of IgA antibodies against some antigens expressed in renal glomeruli in gddY mouse serum. Next, we confirmed this result by WB analysis using glomerular proteins isolated from gddY mouse kidneys. Serum IgA from gddY mice bound to several glomerular proteins, with the highest frequency of IgA antibodies detected against a protein with a molecular weight of approximately 250 kDa (referred to as p250+) (Figure 1B). Next, because IgA molecules are deposited in the mesangial region of renal glomeruli in IgA nephropathy, we performed WB analysis using primary cultured MCs from Balb / c mouse kidneys. Serum IgA from gddY mice prominently detected p250+ bands (Figure 1C), demonstrating that p250+ is the primary target antigen for IgA autoantibodies in gddY mouse serum. Seven of 10 gddY mice had serum IgA that recognized p250+, compared with only one of 16 Balb / c mice (Figure 1D).

[0046] (2) Spectrin β is the target antigen of IgA autoantibodies in gddY mice. To identify the autoantigen recognized by serum IgA autoantibodies from gddY mice, immunoprecipitation of mouse glomerular proteins was performed using serum from gddY mice or Balb / c mice. The precipitated proteins were separated by SDS-PAGE, and proteins precipitated only by gddY mouse serum were analyzed by mass spectrometry. Expression vectors for the candidate autoantigens identified were constructed and transfected into HEK293T cells. Autoantigen screening was performed by Western Blot analysis using cell lysates and gddY serum. Spectrin β-chain non-erythroid 1 (Sptbn1) was identified as a protein recognized exclusively by serum IgA from gddY mice. Next, we narrowed down the region recognized by the autoantibody. Therefore, we divided Sptbn1 into three regions (Sptbn1A: 1-788aa; Sptbn1B: 789-1576aa; Sptbn1C: 577-2364aa), separately expressed each fragment in HEK293T cells, and performed WB analysis. The Sptbn1C fragment was specifically recognized by serum IgA from gddY mice (Figure 2B-D). This assay revealed that 12 of 20 gddY mice had serum IgA autoantibodies against Sptbn1C (Figure 2E). Among more than 10 other candidate proteins tested in the same WB assay, only the C-terminal fragment of spectrin α-chain non-erythroid 1 (Sptan1C) was selectively recognized by serum IgA from gddY mice. However, the frequency of anti-Sptan1C IgA antibodies in gddY mice was much lower (3 of 22) than that of anti-Sptbn1 IgA antibodies, suggesting that Sptbn1 is the major target antigen for IgA autoantibodies in gddY mice.

[0047] (3) Patients with IgA nephropathy have IgA autoantibodies against antigens expressed on mesangial cells The possibility of the presence of IgA-type autoantibodies against MC in the sera of IgA nephropathy patients was examined by WB using proteins extracted from human MC cells. The results showed that the sera of most IgA nephropathy patients contained IgA that recognized p250+ (85%). In contrast, the positive rate of this IgA antibody in the sera of healthy individuals was much lower, at 13% (Fig. 3A, B). Based on the similarity in size of the p250+ protein recognized by sera from IgA nephropathy patients and gddY mice, we hypothesized that serum IgA from IgA nephropathy patients recognizes human SPTBN1. Therefore, we performed WB analysis using patient sera and HEK293T cells infected with a mock vector or a vector expressing human SPTBN1 (full-length). Results showed that 15 of 26 patients with IgA nephropathy had anti-SPTBN1 IgA1 antibodies. In contrast, serum anti-SPTBN1 IgA1 antibodies were not detected in any serum of healthy controls, and were only detected in 2 of 21 patients with other renal diseases (Figures 3C and 3D). These findings demonstrate the presence of IgA1 autoantibodies against SPTBN1 specifically in patients with IgA nephropathy.

[0048] (4) IgA-positive plasmablast cells accumulate in the kidneys of gddY mice. The above results strongly suggest that IgA nephropathy is an autoimmune disease with tissue-specific autoantibodies. Recently, it has been reported that autoantibody-secreting plasma cells are present in inflamed tissues in several autoimmune diseases. Based on this, we hypothesized that autoantibody-producing plasma cells infiltrate the gddY kidney. Therefore, we analyzed mononuclear cells isolated from the kidneys of gddY mice by flow cytometry. Balb / c mice, lupus model mice (NZB / w F1 and Fas lpr / lpr) were used as a comparison. Results revealed that IgA+CD138+ plasma cells significantly infiltrated the kidneys of gddY mice compared with other mice (Fig. 4A, C, D). These IgA+CD138+ cells, like germinal center B cells, were positive for the proliferation marker Ki67 (Fig. 4B, Fig. 8), suggesting that they were short-lived plasmablasts (PBs). The frequency of IgA+ PBs gradually increased with age in gddY mice (Fig. 4E). Immunofluorescent staining of the kidneys of 8wgddY mice confirmed the presence of IgA+ PBs in the tubulointerstitium (Fig. 4F). Consistent with previous reports, IgG was the predominant subclass of plasma cells infiltrating the kidneys of NZB / w F1 mice (Fig. 9), whereas the plasmablast subclass in the kidneys of gddY mice was predominantly IgA (Fig. 4G).

[0049] (5) IgA-secreting cells are present in the kidneys of patients with IgA nephropathy. Next, we investigated whether IgA antibody-secreting cells (ASCs) are also present in the kidneys of patients with IgA nephropathy. Immunochemical staining of kidney biopsy sections from patients with IgA revealed IgA-positive ASCs in the tubulointerstitial region of the kidney, as in gddY mice (Fig. 5A). Similar to reports in patients with SLE, the number of infiltrating ASCs correlated with serum creatinine levels and proteinuria in patients with IgA nephropathy (Fig. 5B, C).

[0050] (6) IgA+PBs infiltrating the kidneys of gddY mice produce IgA autoantibodies. Next, we investigated the possibility that IgA+ PBs infiltrating the kidneys of gddY mice produce IgA-type autoantibodies. We isolated and cultured these cells from gddY kidneys (Fig. 6A), and performed immunofluorescence staining and WB analysis using the culture supernatants. The results revealed that IgA contained in these culture supernatants bound to glomerular components, particularly p250+ proteins (Fig. 6B, C). Furthermore, we confirmed that these IgAs recognized Sptbn1C (Fig. 6D). To confirm that IgA+ PBs in the kidneys of gddY mice produce IgA-type autoantibodies against antigens in MCs, we isolated a single IgA+ PB from the kidney of a gddY mouse, cloned its heavy and light chain genes, and produced recombinant IgA antibodies. Most of the H and L chains contained a significant number of mutations in their amino acid sequences, indicating that the PBs had undergone somatic hypermutation (Fig. 10). Using these recombinant antibodies as primary antibodies, we performed intracellular staining of MCs and analyzed them by flow cytometry. Although the staining intensity varied among the antibodies, 8 of the 20 recombinant antibodies recognized antigens present in MCs (Fig. 6E, F). Furthermore, WB confirmed that five of the 20 recombinant antibodies recognized Sptbn1C (Fig. 6g, H). These results demonstrated that gddY mice, like other animal models and patients with autoimmune diseases, exhibit pathological features characteristic of autoimmune diseases (infiltration of autoantibody-producing ASCs into inflamed tissues).

[0051] (7) Sptbn1, an intracellular protein, is presented on the cell surface by MHC class II molecules. Next, we explored the mechanism by which the intracellular protein Sptbn1 is recognized by anti-spectrin β antibodies in the serum of gddY mice. Recently, it has been reported that misfolded intracellular proteins are transported to the cell surface by binding to MHC class II molecules, which are aberrantly expressed on tissue cells, and that these proteins can serve as specific targets for autoantibodies. MCs are known to express MHC class II both in vivo and in vitro, and MHC class II expression has been reported in renal glomeruli of patients with IgA nephropathy. Based on this, we hypothesized that intracellular Sptbn1 is presented on the surface of MCs by binding to MHC class II molecules. First, we confirmed the expression of MHC class II (IA) on the surface of MCs from gddY mice stimulated with IFN-γ by flow cytometry (Figure 11). Next, we analyzed the binding of recombinant antibodies generated from IgA+PBs in gddY kidneys to the surface of IFN-γ-stimulated MCs by flow cytometry. As a result, WB revealed that the recombinant antibody clone #9, which recognizes the MC antigen (Sptbn1C (Figure 6G)), bound to the surface of IA-positive MCs (Figure 7A). To verify whether Sptbn1 can be presented on the cell surface by MHC class II, we established HEK293T cell lines (IA HEK) that constitutively express IA by retroviral infection with vectors expressing the HA-tagged α chain and FLAG-tagged β chain derived from gddY (H-2S) or C57BL / 6 (H-2b) mice. The expression of IA on the cell surface of IA HEK cells was confirmed by flow cytometry (Fig. 12). Next, we overexpressed a T7-tagged Sptbn1C expression vector in IA HEK cells and analyzed their surface expression by flow cytometry. The results demonstrated that Sptbn1C was presented on the cell surface of IA HEK cells regardless of the IA haplotype (Fig. 7B). These results were confirmed by WB analysis. IA HEK cells were infected with T7-tagged Sptbn1C, and MHC class II proteins were immunoprecipitated from cell lysates using anti-FLAG antibodies. Immunoblotting of the precipitated proteins with anti-T7 antibodies revealed that Sptbn1C co-precipitated with IA molecules (Fig. 7C), confirming the association of Sptbn1C and IA molecules. These results suggest that the intracellular protein Sptbn1C is presented on the surface of MCs by binding to MHC class II molecules, and is thereby recognized by anti-spectrin β IgA antibodies in the serum of gddY mice.

Claims

1. A method for measuring an autoantibody associated with IgA nephropathy, comprising measuring the amount of anti-spectrin β IgA antibody in a biological sample.

2. A diagnostic agent for IgA nephropathy containing a reagent for measuring anti-spectrin β IgA antibody.

3. 3. The diagnostic agent for IgA nephropathy according to claim 2, wherein the reagent for measuring anti-spectrin β IgA antibody is an immunological reagent containing spectrin β.

4. A method for screening for a preventive or therapeutic agent for IgA nephropathy, comprising screening for a drug that inhibits the activity or production of anti-spectrin β IgA antibody.

5. A preventive or therapeutic drug for IgA nephropathy, which comprises as an active ingredient a drug that inhibits the activity or production of anti-spectrin β IgA antibody.

6. A drug that inhibits the activity or production of anti-spectrin β IgA antibodies, used to prevent or treat IgA nephropathy.

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