Method for acquiring information on effectiveness of chemotherapy for membranous nephropathy patient, method for assisting determination of effectiveness of chemotherapy for membranous nephropathy patient, and reagent kit

Abstract

To provide means for enabling determination of the effectiveness of chemotherapy using an anti-CD20 antibody drug for a membranous nephropathy patient.SOLUTION: A method includes measuring CXCL13 in a biological sample collected from a membranous nephropathy patient, and using a measurement result as an index for suggesting the effectiveness of chemotherapy using an anti-CD20 antibody drug for the patient.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for obtaining information on the efficacy of chemotherapy for patients with membranous nephropathy. The present invention relates to a method for assisting in determining the efficacy of chemotherapy for patients with membranous nephropathy. The present invention relates to a reagent kit for use in these methods. [Background technology]

[0002] Membranous nephropathy is a type of glomerulonephritis and is most frequently diagnosed in patients with nephrotic syndrome. Nephrotic syndrome is a clinical condition characterized by edema, proteinuria, decreased plasma albumin, and increased blood cholesterol. In membranous nephropathy, the glomerular basement membrane thickens due to the deposition of immune complexes consisting of the causative antigen and the corresponding autoantibody on the epithelial side of the basement membrane. As a result, the filtration function of the glomerulus decreases, causing chronic proteinuria and edema. Approximately 80% of membranous nephropathy is primary membranous nephropathy (also called idiopathic membranous nephropathy) of unknown cause, and the remaining approximately 20% is secondary membranous nephropathy (also called secondary membranous nephropathy) associated with other diseases. Causes of secondary membranous nephropathy include autoimmune diseases, infections, malignant tumors, and drugs. In recent years, it has been reported that in approximately 70% of cases of primary membranous nephropathy, the causative antigen is the M-type phospholipase A2 receptor (PLA2R) expressed in glomerular epithelial cells. Currently, testing for the amount of anti-PLA2R antibodies in serum is used to classify the risk of membranous nephropathy and determine treatment options.

[0003] Conventionally, in the treatment of membranous nephropathy, steroids were administered alone, and if the therapeutic effect was insufficient, a combination therapy of steroids and immunosuppressants was performed. In recent years, it has been reported that chemotherapy using the anti-CD20 antibody drug rituximab had a good therapeutic effect on patients with refractory nephrotic syndrome due to membranous nephropathy. Non-Patent Document 1 reports a study (STARMEN study) that verifies the hypothesis that sequential administration of tacrolimus and rituximab is superior to alternating administration of corticosteroids and cyclophosphamide. In this study, verification was performed on patients with primary membranous nephropathy and persistent nephrotic syndrome. As a result, the number of patients who benefited from chemotherapy using rituximab was fewer than that of patients who benefited from alternating administration of corticosteroids and cyclophosphamide. However, chemotherapy using rituximab was effective in about half of the patients who received chemotherapy. Therefore, chemotherapy using anti-CD20 antibody drugs is expected to be a promising treatment for membranous nephropathy. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Fernadez-Juarez G. et al., The STARMEN trial indicates that alternating treatment with corticosteroids and cyclophosphamide is superior to sequential treatment with tacrolimus and rituximab in primary membranous nephropathy, Kidney International, vol.99, pp.986-998, 2021 Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, chemotherapy using anti-CD20 antibody drugs is not effective for all membranous nephropathy patients. Anti-CD20 antibody drugs are expensive and may cause serious side effects. Therefore, it is necessary to administer anti-CD20 antibody drugs to patients who are expected to respond. However, there is no known means for predicting the effectiveness of chemotherapy using anti-CD20 antibody drugs for membranous nephropathy patients in advance. The present invention aims to provide a means for determining in advance whether chemotherapy using anti-CD20 antibody drugs will be effective for membranous nephropathy patients. [Means for solving the problem]

[0006] The present inventors have discovered CXCL13 (CXC chemokine ligand 13) as a biomarker associated with the efficacy of chemotherapy using anti-CD20 antibody drugs for patients with membranous nephropathy, and have completed the present invention. Accordingly, the following inventions [1] to

[12] are provided.

[0007] [1] A method for obtaining information regarding the efficacy of chemotherapy for a patient with membranous nephropathy, comprising the step of measuring CXCL13 in a biological sample collected from the patient, the measurement results indicating the efficacy of chemotherapy using an anti-CD20 antibody drug for the patient.

[0008] [2] The method according to the above [1], wherein a measurement of CXCL13 below a threshold level indicates that the patient will respond to chemotherapy, and a measurement of CXCL13 above the threshold level indicates that the patient will not respond to chemotherapy.

[0009] [3] The method according to the above [1] or [2], wherein the anti-CD20 antibody drug is at least one selected from the group consisting of rituximab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab and veltuzumab.

[0010] [4] The method according to any one of the above [1] to [3], wherein the biological sample is a blood sample or urine sample.

[0011] [5] The method according to any one of the above [1] to [4], wherein the chemotherapy uses other drugs in addition to the anti-CD20 antibody drug.

[0012] [6] The method according to [5] above, wherein the other drug is at least one selected from the group consisting of immunosuppressants and steroids.

[0013] [7] The method according to the above [6], wherein the immunosuppressant is a calcineurin inhibitor.

[0014] [8] The method according to the above [7], wherein the calcineurin inhibitor is at least one selected from the group consisting of tacrolimus, pimecrolimus, cyclosporine and voclosporin.

[0015] [9] The method according to the above [6], wherein the steroid is a corticosteroid.

[0016]

[10] A method for assisting in determining the efficacy of chemotherapy for a patient with membranous nephropathy, comprising the steps of measuring CXCL13 in a biological sample collected from the patient and determining the efficacy of chemotherapy using an anti-CD20 antibody drug for the patient based on the measurement results.

[0017]

[11] The method according to the above

[10] , wherein the patient is judged to be effective against chemotherapy when the measured value of CXCL13 is lower than a threshold value, and the patient is judged to be ineffective against chemotherapy when the measured value of CXCL13 is equal to or higher than the threshold value.

[0018]

[12] A reagent kit for use in the method according to any one of [1] to

[11] above, comprising a reagent containing a substance capable of specifically binding to CXCL13. Effect of the Invention

[0019] According to the present invention, it is possible to determine whether chemotherapy using an anti-CD20 antibody drug will be effective for a patient with membranous nephropathy. [Brief description of the drawings]

[0020] [Figure 1] FIG. 2 is a diagram showing an example of the appearance of a reagent kit. [Diagram 2] 1 is a box plot showing the concentration of CXCL13 (uCXCL13) in urine samples in chemotherapy responders and non-responders. [Diagram 3] 1 is a box plot showing the concentration of IL-17 (uIL17) in urine samples in chemotherapy responders and non-responders. [Figure 4] 1 is a box plot showing the concentration of TWEAK in urine samples (uTWEAK) in chemotherapy responders and non-responders. [Diagram 5] 1 is a box plot showing the concentration of GDF15 (uGDF15) in urine samples in chemotherapy responders and non-responders. [Figure 6] 1 is a box plot showing the concentration of TNF-α (uTNF-α) in urine samples in chemotherapy responders and non-responders. [Figure 7] 1 is a box plot showing serum CXCL13 concentrations in chemotherapy responders and non-responders. [Figure 8] 1 is a box plot showing serum IL-17 concentrations in chemotherapy responders and non-responders. [Figure 9] 1 is a box plot showing serum TWEAK concentrations in chemotherapy responders and non-responders. [Figure 10] 1 is a box plot showing serum GDF15 concentrations in chemotherapy responders and non-responders. [Figure 11] 1 is a box plot showing serum TNF-α concentrations in chemotherapy responders and non-responders. [Figure 12] 1 is a box plot showing serum MIG concentrations in chemotherapy responders and non-responders. [Figure 13] 1 is a box plot showing serum CCL20 concentrations in chemotherapy responders and non-responders. [Figure 14]1 is a box plot showing serum anti-PLA2R antibody concentrations in chemotherapy responders and non-responders. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In a method for obtaining information regarding the efficacy of chemotherapy for a patient with membranous nephropathy, CXCL13 is measured in a biological sample collected from the patient with membranous nephropathy.

[0022] In this specification, the term "membranous nephropathy patients" includes both patients diagnosed with membranous nephropathy and those suspected of having membranous nephropathy. Membranous nephropathy may be either primary membranous nephropathy or secondary membranous nephropathy. Preferably, the "membranous nephropathy patients" are membranous nephropathy patients who have not yet undergone chemotherapy using an anti-CD20 antibody drug. More preferably, the "membranous nephropathy patients" are membranous nephropathy patients who are subject to immunosuppressive treatment and have not yet undergone chemotherapy using an anti-CD20 antibody drug. Immunosuppressive treatment refers to treatment using an immunosuppressant drug.

[0023] The diagnosis of membranous nephropathy is made by diagnosis of associated pathologies, serum anti-PLA2R antibody test and kidney biopsy, which are definitive diagnoses. The diagnosis of associated pathologies is made by, for example, X-ray or CT scan to diagnose the presence or absence of sarcoidosis, drug history such as nonsteroidal anti-inflammatory drugs (NSAIDs), gold preparations and penicillamine, antinuclear antibody test, malignant tumor screening, renal ultrasound, diagnosis of hepatitis B, hepatitis C, HIV and treponemal infection, disease history such as thyroid disease and systemic disease, and physical examination of the skin and connective tissue.

[0024] Chemotherapy using an anti-CD20 antibody drug refers to a method of treating a disease using at least an anti-CD20 antibody drug as a drug. An anti-CD20 antibody drug refers to a drug that contains an antibody that specifically binds to CD20 and exhibits the action of damaging and removing CD20-positive cells. It is known that CD20 is specifically expressed on the surface of differentiating B cells and B cell lymphoma cells. The antibody contained in the anti-CD20 antibody drug is preferably a chimeric (human / mouse) monoclonal antibody or a fully human monoclonal antibody. The antibody contained in the anti-CD20 antibody drug may be labeled with a pharma- ceutical acceptable radioactive substance such as iodine-131. The anti-CD20 antibody drug itself is known, and examples thereof include rituximab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, and veltuzumab.

[0025] In chemotherapy using an anti-CD20 antibody drug, other drugs may be used in addition to the anti-CD20 antibody drug. Examples of other drugs include immunosuppressants and steroids. Examples of immunosuppressants include calcineurin inhibitors, alkylating agents, purine metabolic antagonists, and mTOR inhibitors. Examples of calcineurin inhibitors include tacrolimus, pimecrolimus, cyclosporine, and voclosporin. Examples of alkylating agents include cyclophosphamide and chlorambucil. Examples of purine metabolic antagonists include mizoribine, mycophenolate mofetil, and azathioprine. Examples of mTOR inhibitors include everolimus, sirolimus, and temsirolimus. Among them, calcineurin inhibitors are preferred. Examples of steroids include corticosteroids, such as prednisolone.

[0026] CXCL13 is a type of chemokine, also called BCA-1 (B cell attracting chemokine 1) or BLC (B lymphocyte chemoattractant). CXCL13 is known as a protein ligand for the CXCR5 receptor expressed on the surface of B cells and follicular T cells. The amino acid sequence of CXCL13 itself is publicly known and is disclosed in public databases such as NCBI (National Center for Biotechnology Information).

[0027] The biological sample is not particularly limited as long as it is a sample containing CXCL13. Examples of such biological samples include blood samples, urine, cerebrospinal fluid, pleural effusion, ascites, and lymph. Examples of blood samples include blood (whole blood) collected from a subject, and plasma or serum prepared from the blood. When the biological sample contains insoluble impurities such as cells, the impurities may be removed from the biological sample by known means such as centrifugation and filtration. The biological sample may be diluted with an appropriate aqueous medium as necessary. Such an aqueous medium is not particularly limited as long as it does not interfere with the measurement described below, and examples of the aqueous medium include water, saline, and buffer solutions. The buffer solution is not particularly limited as long as it has a buffering effect at a pH near neutral (for example, a pH of 6 to 8). Examples of such buffer solutions include Good's buffer solutions such as HEPES, MES, and PIPES, phosphate buffered saline (PBS), and Tris buffered saline (TBS).

[0028] As used herein, "measuring CXCL13" includes determining the amount or concentration of CXCL13 and obtaining information reflecting the amount or concentration of CXCL13. "Information reflecting the amount or concentration of CXCL13" refers to an indicator that changes depending on the amount or concentration of CXCL13 in a biological sample or a measurement sample prepared from the biological sample. Such an indicator is preferably an indicator of optical change that is visible or measurable by a machine. Examples of indicators of optical change include luminescence intensity, fluorescence intensity, absorbance, turbidity, and color intensity.

[0029] The information reflecting the amount or concentration of CXCL13 may be shown qualitatively, quantitatively, or semi-quantitatively. Qualitatively shown information is information showing the presence or absence of CXCL13. Quantitatively shown information is numerical information such as a value obtained by a measuring device (hereinafter also referred to as "raw data") and a value calculated from the numerical value. Examples of values ​​calculated from raw data include a value obtained by subtracting a negative control sample value or a background value from the raw data. The amount or concentration value of CXCL13 can be determined based on the quantitative information. Semi-quantitatively shown information is information showing the amount or concentration of CXCL13 in a graded manner using words, numbers (indicating a rank), colors, etc. For example, words such as "below the detection limit", "low", "moderate", and "high" may be used.

[0030] As used herein, the term "measurement result of CXCL13" includes values, information, and combinations thereof obtained by measuring CXCL13. Preferably, the measurement result of CXCL13 is quantitative information reflecting the amount or concentration of CXCL13, and / or a value of the amount or concentration of CXCL13 determined based on the quantitative information. Hereinafter, the quantitative information reflecting the amount or concentration of CXCL13 and / or the value of the amount or concentration of CXCL13 is also referred to as "measurement value of CXCL13".

[0031] The method for measuring CXCL13 is not particularly limited, as long as it can obtain information reflecting the amount or concentration of CXCL13 in a biological sample or a measurement sample prepared from a biological sample. For example, such a method includes a method of capturing CXCL13 using a substance that can specifically bind to CXCL13. CXCL13 captured by such a substance can be detected by a known method to measure CXCL13 in a biological sample.

[0032] Substances capable of specifically binding to CXCL13 include, for example, antibodies, aptamers, receptor proteins, and the like. Among them, antibodies are particularly preferred. Antibodies against CXCL13 are known and generally available. There are no particular limitations on the antibodies against CXCL13, so long as they can specifically bind to CXCL13. Such antibodies may be any of monoclonal antibodies, polyclonal antibodies, and fragments thereof (e.g., Fab, F(ab')2, Fab', etc.). Commercially available antibodies may also be used.

[0033] The method of measuring CXCL13 using an antibody is not particularly limited and can be appropriately selected from known immunological measurement methods. Examples of such measurement methods include enzyme-linked immunosorbent assay (ELISA) and Western blot. Among them, ELISA is preferred. The type of ELISA may be any of sandwich, competitive, direct, and indirect methods, but sandwich is particularly preferred. As an example, the measurement of CXCL13 in a biological sample by sandwich ELISA will be described below.

[0034] First, a complex containing CXCL13, an antibody for capturing CXCL13 (hereinafter also referred to as a "capture antibody"), and an antibody for detecting CXCL13 (hereinafter also referred to as a "detection antibody") is formed on a solid phase. This complex can be formed by mixing a biological sample that may contain CXCL13, the capture antibody, and the detection antibody. Then, the above complex can be formed on the solid phase by contacting a solution containing the complex with a solid phase that can capture the capture antibody. Alternatively, a solid phase on which the capture antibody is immobilized in advance may be used. That is, the above complex can be formed on the solid phase by contacting a solid phase on which the capture antibody is immobilized with a biological sample and a detection antibody. When both the capture antibody and the detection antibody are monoclonal antibodies, it is preferable that they have different epitopes.

[0035] The solid phase may be an insoluble carrier capable of immobilizing the capture antibody. The manner of immobilization of the capture antibody to the solid phase is not particularly limited. For example, the capture antibody may be directly bound to the solid phase, or the capture antibody may be indirectly bound to the solid phase via another substance. An example of direct binding is physical adsorption. An example of indirect binding is binding via a combination of biotins and avidins. The capture antibody is modified with biotins in advance, and avidins are bound to the solid phase in advance, so that the capture antibody can be indirectly bound to the solid phase via the binding between the biotins and the avidins.

[0036] As used herein, "biotins" include biotin and its analogues. Examples of biotin analogues include desthiobiotin and biocytin. As used herein, "avidins" include avidin and its analogues. Examples of avidin analogues include streptavidin, avidin-like protein derived from Pleurotus cornucopiae (Tamavidin (registered trademark)), bladdavidin, and resavidin.

[0037] The material of the solid phase is not particularly limited, and can be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, etc. Examples of organic polymer compounds include latex, polystyrene, polypropylene, etc. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, etc. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, cellulose, etc. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples include particles, membranes, microplates, microtubes, test tubes, etc. Among these, particles are preferred, and magnetic particles are particularly preferred.

[0038] Between the complex formation step and the complex detection step, B / F (Bound / Free) separation may be performed to remove unreacted free components that do not form a complex. The unreacted free components refer to components that do not form a complex. Examples include capture antibodies and detection antibodies that have not bound to CXCL13. There is no particular limitation on the means of B / F separation. If the solid phase is a particle, B / F separation can be performed by recovering only the solid phase that has captured the complex by centrifugation. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing the unreacted free components. In addition, if the solid phase is a magnetic particle, B / F separation can be performed by aspirating and removing the liquid containing the unreacted free components with a nozzle while the magnetic particles are magnetically restrained by a magnet, which is preferable from the viewpoint of automation. After removing the unreacted free components, the solid phase that has captured the complex may be washed with an appropriate aqueous medium such as PBS.

[0039] The complex formed on the solid phase can be detected by a known method to measure CXCL13 in a biological sample. For example, when an antibody labeled with a labeling substance is used as the detection antibody, a signal due to the labeling substance is generated from the detection antibody contained in the complex on the solid phase. The complex on the solid phase is detected by detecting the signal. Since this complex contains CXCL13 captured from the biological sample by the capture antibody, CXCL13 in the biological sample is measured by detecting the complex. When a labeled secondary antibody against the detection antibody is used, CXCL13 in the biological sample can be measured in the same manner.

[0040] As a further example of a method for measuring CXCL13 using an antibody, the immune complex transfer method described in JP-A-1-254868 can also be used.

[0041] The labeling substance is not particularly limited as long as it generates a detectable signal. For example, it may be a substance that generates a signal by itself (hereinafter also referred to as a "signal generating substance"), or a substance that generates a signal by catalyzing the reaction of other substances. Examples of signal generating substances include fluorescent substances and radioisotopes. Examples of substances that catalyze the reaction of other substances to generate a detectable signal include enzymes. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, luciferase, etc. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioisotopes include 125 I, 14 C. 32 Among them, enzymes are preferred as labeling substances, and alkaline phosphatase and peroxidase are particularly preferred.

[0042] The method for detecting a signal is known in the art. A measurement method can be appropriately selected according to the type of signal derived from the labeling substance. For example, when the labeling substance is an enzyme, the signal such as light or color generated by reacting with a substrate for the enzyme can be measured using a known device such as a spectrophotometer.

[0043] The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when alkaline phosphatase is used as the enzyme, the substrate can be a chemiluminescent substrate such as CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), or a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, or p-nitrophenyl phosphate. When peroxidase is used as the enzyme, examples of the substrate include chemiluminescent substrates such as luminol and its derivatives, and color-developing substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB).

[0044] When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined depending on the type of the fluorescent substance used.

[0045] The detection result of the signal can be used as the measurement result of CXCL13. For example, when quantifying the signal intensity, the measured value of the signal intensity itself or a value obtained from the measured value can be used as the measurement result of CXCL13. Examples of the value obtained from the measured value of the signal intensity include a value obtained by subtracting the measured value of the negative control sample or the background value from the measured value of the signal intensity. In addition, the measured value of the signal intensity may be applied to a calibration curve to determine the amount or concentration of CXCL13. The negative control sample can be appropriately selected, and examples thereof include a biological sample obtained from a healthy individual.

[0046] CXCL13 contained in a biological sample can be measured by a sandwich ELISA method using a capture antibody immobilized on magnetic particles and a detection antibody labeled with a labeling substance. In this case, the measurement may be performed using a commercially available fully automated immunoassay device. Examples of such fully automated immunoassay devices include the HISCL (registered trademark) series from Sysmex Corporation.

[0047] The measurement result of CXCL13 is an indicator of the efficacy of chemotherapy using anti-CD20 antibody drugs for patients with membranous nephropathy. For example, as shown in the examples, the concentration of CXCL13 in the biological sample was significantly lower in the patient group for whom rituximab was effective than in the patient group for whom rituximab was not effective. Therefore, the measurement result of CXCL13 can be obtained as information on the efficacy of chemotherapy using anti-CD20 antibody drugs for patients with membranous nephropathy. For example, when the measurement value of CXCL13 is lower than the threshold value, the measurement value of CXCL13 can suggest that chemotherapy using anti-CD20 antibody drugs is effective for patients with membranous nephropathy. When the measurement value of CXCL13 is equal to or higher than the threshold value, the measurement value of CXCL13 can suggest that chemotherapy using anti-CD20 antibody drugs is not effective for patients with membranous nephropathy.

[0048] Alternatively, when the measured value of CXCL13 is equal to or lower than the threshold, the measured value of CXCL13 may suggest that chemotherapy using an anti-CD20 antibody drug is effective for a patient with membranous nephropathy.When the measured value of CXCL13 is higher than the threshold, the measured value of CXCL13 may suggest that chemotherapy using an anti-CD20 antibody drug is not effective for a patient with membranous nephropathy.

[0049] The threshold for the measured value of CXCL13 is not particularly limited and can be set appropriately. For example, the threshold may be set as follows. First, biological samples are collected from a plurality of membranous nephropathy patients who responded to chemotherapy using an anti-CD20 antibody drug (response group) and a plurality of membranous nephropathy patients who did not respond to the chemotherapy (non-response group), and CXCL13 is measured to obtain a measured value of CXCL13. Then, a value that can most accurately distinguish between the response group and the non-response group is obtained, and this value is set as the threshold. In setting the threshold, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, etc.

[0050] The present invention may include a step of determining the efficacy of chemotherapy using an anti-CD20 antibody drug for a patient with membranous nephropathy based on the measurement result. Preferably, the measured value of CXCL13 is compared with a threshold value, and a determination is made based on the comparison result. For example, when the measured value of CXCL13 is lower than the threshold value, it may be determined that chemotherapy using an anti-CD20 antibody drug is effective for a patient with membranous nephropathy. When the measured value of CXCL13 is equal to or higher than the threshold value, it may be determined that chemotherapy using an anti-CD20 antibody drug is ineffective for a patient with membranous nephropathy. Alternatively, when the measured value of CXCL13 is equal to or lower than the threshold value, it may be determined that chemotherapy using an anti-CD20 antibody drug is effective for a patient with membranous nephropathy. When the measured value of CXCL13 is higher than the threshold value, it may be determined that chemotherapy using an anti-CD20 antibody drug is ineffective for a patient with membranous nephropathy. The details of the patient with membranous nephropathy, chemotherapy using an anti-CD20 antibody drug, biological sample, measurement of CXCL13, and threshold value are as described above.

[0051] The present invention also includes a method for treating a patient with membranous nephropathy based on the above-mentioned determination result. The treatment method includes the steps of measuring CXCL13 in a biological sample collected from a patient with membranous nephropathy, determining the efficacy of chemotherapy using an anti-CD20 antibody drug for the patient based on the measurement result of CXCL13, and performing chemotherapy using an anti-CD20 antibody drug on the patient when the chemotherapy is determined to be effective for the patient. In the determination step, when the measured value of CXCL13 is lower than a threshold, it is determined that chemotherapy using an anti-CD20 antibody drug is effective for the patient with membranous nephropathy. Alternatively, when the measured value of CXCL13 is equal to or lower than the threshold, it is determined that chemotherapy using an anti-CD20 antibody drug is effective for the patient with membranous nephropathy. The details of the patient with membranous nephropathy, chemotherapy using an anti-CD20 antibody drug, biological sample, measurement of CXCL13, and threshold are as described above.

[0052] In the chemotherapy step, it is preferable to administer an effective amount of an anti-CD20 antibody drug to the patient. In addition to the anti-CD20 antibody drug, other immunosuppressants may be further administered. The effective amount can be appropriately determined according to the treatment guidelines for membranous nephropathy, etc.

[0053] The present invention also includes a reagent kit (hereinafter also referred to as "reagent kit") used in a method for obtaining information on the efficacy of chemotherapy for patients with membranous nephropathy, or a method for assisting in the assessment of the efficacy of chemotherapy for patients with membranous nephropathy. The reagent kit includes a reagent containing a substance capable of specifically binding to CXCL13. Details of the substance capable of specifically binding to CXCL13 are as described above.

[0054] The reagent containing a substance capable of specifically binding to CXCL13 is preferably a combination of a reagent containing a capture antibody against CXCL13 and a reagent containing a detection antibody against CXCL13. The reagent kit contains a reagent containing a capture antibody against CXCL13 and a reagent containing a detection antibody against CXCL13. The detection antibody may be labeled with a labeling substance. When the labeling substance is an enzyme, the reagent kit may contain a substrate for the enzyme. Details of the capture antibody, detection antibody, labeling substance, and substrate are as described above. The forms of the capture antibody, detection antibody, labeling substance, and substrate are not particularly limited, and may be solid (e.g., powder, crystal, freeze-dried product, etc.) or liquid (e.g., solution, suspension, emulsion, etc.).

[0055] The reagent kit may be provided to a user in a state where a container containing a reagent is packed in a box. The box may include a package insert. The package insert may describe the configuration of the reagent kit, the method of use, the relationship between the measurement results obtained by the reagent kit and the pathology of a patient with membranous nephropathy, and the like. An example of such a reagent kit is shown in FIG. 1. The reagent kit shown in FIG. 1 includes a reagent containing a capture antibody against CXCL13 and a reagent containing a detection antibody against CXCL13, but the present invention is not limited to this example. With reference to FIG. 1, 11 indicates the reagent kit, 12 indicates a first container containing a reagent containing a capture antibody against CXCL13, 13 indicates a second container containing a reagent containing a detection antibody against CXCL13, 14 indicates a packaging box, and 15 indicates the package insert. In this example, the reagent kit may further include a solid phase for immobilizing the capture antibody against CXCL13. The details of the solid phase are as described above.

[0056] The reagent kit may further include a calibrator for quantification of CXCL13. The calibrator for quantification of CXCL13 is, for example, a buffer solution containing a recombinant CXCL13 protein at a predetermined concentration. The number of buffer solutions containing CXCL13 at a predetermined concentration may be one or more. When there are multiple buffer solutions containing CXCL13 at a predetermined concentration, it is preferable to prepare each buffer solution by serial dilution or the like so that the CXCL13 concentrations are different from each other. The calibrator for quantification of CXCL13 may further include a buffer solution not containing CXCL13 (negative control).

[0057] The present invention includes the use of a reagent for producing a reagent kit used in a method for assisting in determining the efficacy of chemotherapy for a patient with membranous nephropathy. The details of the reagent are as described above.

[0058] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0059] Example 1 (1) Biological samples As biological samples, urine and blood samples were used from 14 patients with primary membranous nephropathy who were treated with rituximab and tacrolimus in the STARMEN study (see Non-Patent Document 1). These patients were not receiving chemotherapy using rituximab and tacrolimus at the time of urine and blood collection. Urine samples were prepared by diluting the second urine collected from each patient in the morning 4-fold with PBS (containing 1% bovine serum albumin (BSA), 0.05% sodium azide and 50 mM EDTA). Blood samples were serum prepared by a conventional method from blood collected from each patient. These samples were obtained from REDinREN Biobank (Address: Universidad de Alcala, Facultad de Medicina, Carretera Madrid-Barcelona, ​​Km. 33.600, Alcala de Henares, Madrid Spain, Representative: Laura Calleros Basilio).

[0060] (2) Patient information The above patient was an adult with primary membranous nephropathy diagnosed by biopsy and had been observed for at least 6 months. 2 eGFR is 45 mL / min or more (≥45 mL / min / 1.73m 2 ). Proteinuria was greater than 4 g / 24 h (>4 g / 24 h), consistent with nephrotic syndrome. There was no greater than 50% reduction in proteinuria during the observation period. Serum albumin was less than 3.5 g / dL (≦3.5 g / dL), consistent with hypoalbuminemia, during the observation period. The patients received standard treatment (angiotensin-converting enzyme inhibitor / angiotensin receptor blocker therapy) for at least 2 months and blood pressure control (≦150 / 90 mmHg) for at least 3 months.

[0061] (3) Chemotherapy with rituximab and tacrolimus The patient was administered tacrolimus (0.05 mg / kg / day) orally for 6 months. The target blood concentration was 5-7 ng / mL. Rituximab (1 g) was administered intravenously on the 180th day after the start of treatment, and tacrolimus was reduced by 25% per month. Treatment was terminated at the 9th month. In the case of renal dysfunction, the dose of tacrolimus was reduced. Methylprednisolone (100 mg), acetaminophen (1 g), and diphenhydramine (50 mg) were administered to reduce the side effects of rituximab administration. Trimethoprim / sulfamethoxazole (160 / 800 mg) was also administered orally three times a week during the treatment period.

[0062] (4) Classification of chemotherapy responders and non-responders Patients who achieved complete or partial remission of membranous nephropathy by chemotherapy with rituximab and tacrolimus were classified as responders. There were five patients in the responder group. Complete remission was defined as a reduction in proteinuria from baseline to 0.3 g / 24 h or less (≦0.3 g / 24 h) and maintenance of renal function (1.73 m 2 Partial response was defined as a reduction in proteinuria by more than 50% from baseline or a proteinuria level of less than 3.5 g / 24 h and preserved renal function (1.73 m 2 Patients were classified as non-responders if their proteinuria reduction was less than 50% from baseline and if their nephrotic syndrome recurred. There were 9 patients in the non-responder group. Patients with recurrence of nephrotic syndrome were defined as those who had previously experienced partial or complete remission but had recurrence of proteinuria of 3.5 g / 24 hours or more and had an increase in proteinuria of at least 50% from the nadir for three or more consecutive visits.

[0063] (5) Measurement of biomarkers CXCL13, IL-17, TWEAK, GDF15, and TNF-α were measured in urine samples from each patient. In addition, CXCL13, IL-17, TWEAK, GDF15, TNF-α, MIG (also called CXCL9), CCL20 (also called MIP-3α), and anti-PLA2R antibodies were measured in blood samples from each patient. IL-17, TWEAK, GDF15, TNF-α, MIG, and CCL20 were biomarkers that have been reported to be associated with glomerulonephritis and / or autoimmune diseases. Anti-PLA2R antibodies were known as causative antigens of primary membranous nephropathy. Measurements were performed using a fully automated immunoassay device HISCL-5000 (Sysmex Corporation). GDF15 was measured using an ELISA reagent purchased from R&D Systems. Anti-PLA2R antibodies were measured using an ELISA reagent purchased from Euroimmun. Biomarkers other than GDF15 and anti-PLA2R antibodies were measured using the following R1 to R5 reagents. The R1 reagent used to measure biomarkers other than TNF-α was a capture reagent containing an antibody that specifically binds to each biomarker except TNF-α. The R1 reagent used to measure TNF-α was the MES buffer described below. The R2 reagent used to measure biomarkers other than TNF-α was a reagent containing magnetic particles as a solid phase. The R2 reagent used to measure TNF-α was a reagent containing magnetic particles to which an antibody that specifically binds to TNF-α was immobilized. The R3 reagent was a detection reagent containing an ALP-labeled antibody that specifically binds to each biomarker. The R4 reagent was a measurement buffer, and the R5 reagent was a reagent containing a chemiluminescent substrate for ALP. The R4 and R5 reagents were used in common to measure each biomarker.

[0064] (5.1) Reagents for measuring CXCL13 R1 Reagent Biotin-labeled anti-CXCL13 monoclonal antibody (Invitrogen) was dissolved in buffer to prepare R1 reagent. For measuring CXCL13 in urine samples, 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 1% BSA, and 0.5% casein) was used as the buffer. For measuring CXCL13 in blood samples, 100 mM HEPES (pH 7.4) (containing 550 mM NaBr, 0.15 mM EDTA 2Na, and 25 mM EGTA) was used as the buffer.

[0065] R2 Reagent Magnetic particles with streptavidin immobilized on the surface (hereinafter referred to as "STA-bound magnetic particles"; average particle size 2 μm; amount of streptavidin per 1 g of magnetic particles 2.9-3.5 mg) were washed three times with 10 mM HEPES (pH 7.5). The washed STA-bound magnetic particles were added to 10 mM HEPES (pH 7.5) so that the streptavidin concentration was 18-22 μg / ml (STA-bound magnetic particle concentration 0.48-0.52 mg / ml) to prepare R2 reagent.

[0066] R3 Reagent An anti-CXCL13 monoclonal antibody (BioLegend) derived from a clone different from the antibody used in the R1 reagent was labeled with ALP by a conventional method. The resulting ALP-labeled anti-CXCL13 antibody was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl and 1% BSA) to prepare the R3 reagent.

[0067] R4 and R5 Reagents As the R4 reagent, HISCL R4 reagent (Sysmex Corporation), which is a measurement buffer, was used. As the R5 reagent, HISCL R5 reagent (Sysmex Corporation) containing CDP-Star (registered trademark) (Applied Biosystems), which is a chemiluminescent substrate for ALP, was used.

[0068] (5.2) Reagents for measuring IL-17 R1 Reagent Biotin-labeled anti-IL-17 monoclonal antibody (Invitrogen) was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 1% BSA, and 0.5% casein) to prepare R1 reagent.

[0069] R3 Reagent An ALP-labeled anti-IL-17 monoclonal antibody (Invitrogen) derived from a clone different from the antibody used in the R1 reagent was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl and 1% BSA) to prepare an R3 reagent.

[0070] (5.3) TWEAK Measurement Reagents R1 Reagent Anti-human TWEAK monoclonal antibody (R&D Systems) was labeled with biotin by a conventional method. The obtained biotin-labeled anti-TWEAK antibody was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 1% casein, 10 mM EDTA, and 10 mM EGTA) to prepare R1 reagent.

[0071] R3 Reagent An anti-TWEAK monoclonal antibody (Abcam) derived from a clone different from the antibody used in the R1 reagent was labeled with ALP by a conventional method. The resulting ALP-labeled anti-TWEAK antibody was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl and 1% BSA) to prepare the R3 reagent.

[0072] (5.4) TNF-α measurement reagent R1 Reagent As the R1 reagent, 100 mM MES (pH 6.5) (containing 150 mM NaCl and 2% BSA) was prepared.

[0073] R2 Reagent Anti-TNF-α monoclonal antibody (Bio-Techne) was labeled with biotin by a conventional method. The obtained biotin-labeled anti-TNF-α antibody was adsorbed onto sensitized magnetic particles and dissolved in 100 mM MES (pH 6.5) (containing 150 mM NaCl and 2% BSA) to prepare R2 reagent.

[0074] R3 Reagent An anti-TNF-α monoclonal antibody (Bio-Techne) derived from a clone different from that used in the R2 reagent was labeled with ALP by a conventional method. The resulting ALP-labeled anti-TNF-α antibody was dissolved in 100 mM MES (pH 6.5) (containing 150 mM NaCl and 2% BSA) to prepare the R3 reagent.

[0075] (5.5) Reagents for measuring MIG R1 Reagent Anti-MIG monoclonal antibody (RANDOX) was digested with pepsin or IdeS protease by a conventional method to obtain a Fab fragment. The Fab fragment was labeled with biotin by a conventional method. The obtained biotin-labeled Fab fragment was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 1% BSA, and 0.5% casein) to prepare R1 reagent.

[0076] R3 Reagent An anti-MIG monoclonal antibody (RANDOX) derived from a clone different from the antibody used in the R1 reagent was digested with pepsin or IdeS protease by a conventional method to obtain a Fab fragment. The Fab fragment was labeled with ALP by a conventional method. The resulting ALP-labeled Fab fragment was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 1% CHAPS, 1% BSA, and 0.5% casein) to prepare the R3 reagent.

[0077] (5.6) CCL20 measurement reagent R1 Reagent Anti-MIP-3α monoclonal antibody (Abcam) was used as the R1 reagent for measuring CCL20 in urine samples. This antibody was labeled with biotin by a conventional method, and the obtained biotin-labeled antibody was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 25 mM EDTA, 25 mM EGTA, and 0.1% Tween (trademark) 20) to prepare the R1 reagent. Anti-human CCL20 (MIP-3α) monoclonal antibody (BioLegend) was used as the R1 reagent for measuring CCL20 in blood samples. This antibody was labeled with biotin by a conventional method, and the obtained biotin-labeled antibody was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 25 mM EDTA, 25 mM EGTA, and 0.1% Tween (trademark) 20) to prepare the R1 reagent.

[0078] R3 Reagent Anti-human CCL20 (MIP-3α) monoclonal antibody (BioLegend) derived from a clone different from the antibody used in the R1 reagent was labeled with ALP by a conventional method. The resulting ALP-labeled antibody was dissolved in 100 mM triethanolamine (pH 7.4) (containing 150 mM NaCl, 1% BSA, and 0.5% casein) to prepare the R3 reagent.

[0079] The measurement procedure using the HISCL-5000 was as follows. For biomarkers other than TNF-α, urine samples were diluted 2- to 4-fold with sample buffer (PBS (pH 7.4) containing 1% BSA and 50 mM EDTA [CXCL13], PBS (pH 7.4) containing 1% casein [TWEAK], PBS (pH 7.4) containing 1% BSA [IL-17]) to prepare samples. After mixing the sample with the R1 reagent, the R2 reagent was added. The magnetic particles in the resulting mixture were collected, the supernatant was removed, and the magnetic particles were washed by adding HISCL washing solution. The supernatant was removed, and the R3 reagent was added to the magnetic particles and mixed. The magnetic particles in the resulting mixture were collected, the supernatant was removed, and the magnetic particles were washed by adding HISCL washing solution. The supernatant was removed, and the R4 and R5 reagents were added to the magnetic particles, and the chemiluminescence intensity was measured. The obtained chemiluminescence intensity was applied to a calibration curve to determine the concentration of the biomarker. Standard curves were constructed using calibrators for each biomarker, which were prepared from recombinant proteins of each biomarker.

[0080] (6) Measurement results The concentrations of CXCL13, IL-17, TWEAK, GDF15 and TNF-α in urine samples from the response group and non-response group are shown in Figures 2 to 6, respectively. The concentrations of CXCL13, IL-17, TWEAK, GDF15, TNF-α, MIG, CCL20 and anti-PLA2R antibody in blood samples from the response group and non-response group are shown in Figures 7 to 14, respectively. In each figure, patients whose biomarkers could not be measured due to insufficient sample volume or other reasons were excluded. Referring to Figure 2, it was found that the concentration of CXCL13 in urine samples from the response group was significantly lower than that of the non-response group (p = 0.017). Referring to Figure 7, it was found that the concentration of CXCL13 in blood samples from the response group was also significantly lower than that of the non-response group (p = 0.020). Therefore, it was shown that CXCL13 in urine and blood can be a biomarker that enables the determination of the efficacy of chemotherapy using anti-CD20 antibody drugs for membranous nephropathy patients. In other words, it was shown that the concentration of CXCL13 in urine or blood samples suggests the efficacy of chemotherapy using anti-CD20 antibody drugs for membranous nephropathy patients. In addition, as can be seen from Figures 2 and 7, it was shown that a threshold value that can distinguish between the response group and the non-response group can be set for the concentration of CXCL13. It was shown that it is possible to determine whether chemotherapy using anti-CD20 antibody drugs is effective for membranous nephropathy patients based on the comparison result between such a threshold value and the concentration of CXCL13 in urine or blood samples.

[0081] Referring to Figures 3 to 6, no significant difference was observed between the response group and the non-response group for IL-17, TWEAK, GDF15, and TNF-α in urine samples. Referring to Figures 8 to 14, no significant difference was observed between the response group and the non-response group for IL-17, TWEAK, GDF15, TNF-α, MIG, CCL20, and anti-PLA2R antibody in blood samples. As described above, these biomarkers have been reported to be associated with glomerulonephritis and / or autoimmune diseases. However, it has been shown that these biomarkers are not associated with the efficacy of chemotherapy using anti-CD20 antibody drugs for membranous nephropathy patients. [Explanation of symbols]

[0082] 11 Reagent Kits 12 1st container 13 Second container 14 Packing box 15 Attached Documents

Claims

1. A method for obtaining information regarding the effectiveness of chemotherapy for a patient with membranous nephropathy, comprising a step of measuring CXCL13 in a biological sample collected from the patient, the measurement results indicating the effectiveness of chemotherapy using an anti-CD20 antibody drug for the patient.

2. The method of claim 1, wherein a measurement of CXCL13 below a threshold indicates that the chemotherapy is effective for the patient, and a measurement of CXCL13 above a threshold indicates that the chemotherapy is not effective for the patient.

3. The method according to claim 1, wherein the anti-CD20 antibody drug is at least one selected from the group consisting of rituximab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab and veltuzumab.

4. The method of claim 1 , wherein the biological sample is a blood sample or urine.

5. The method according to claim 1, wherein the chemotherapy is a chemotherapy using other drugs in addition to the anti-CD20 antibody drug.

6. The method according to claim 5, wherein the other drug is at least one selected from the group consisting of immunosuppressants and steroids.

7. The method of claim 6 , wherein the immunosuppressant is a calcineurin inhibitor.

8. The method according to claim 7, wherein the calcineurin inhibitor is at least one selected from the group consisting of tacrolimus, pimecrolimus, cyclosporine and voclosporin.

9. 7. The method of claim 6, wherein the steroid is a corticosteroid.

10. Measuring CXCL13 in a biological sample collected from a patient with membranous nephropathy; determining the efficacy of chemotherapy using an anti-CD20 antibody drug for the patient based on the measurement results; A method for assisting in determining the efficacy of chemotherapy for a patient with membranous nephropathy, comprising:

11. The method according to claim 10, wherein the chemotherapy is determined to be effective for the patient when the measured value of CXCL13 is lower than a threshold value, and the chemotherapy is determined to be ineffective for the patient when the measured value of CXCL13 is equal to or higher than the threshold value.

12. A reagent kit for use in the method according to any one of claims 1 to 11, comprising a reagent containing a substance capable of specifically binding to CXCL13.

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