Prophylactic or therapeutic agent for disease in which formation of tertiary lymphoid tissue occurs in lesion

Abstract

The present disclosure addresses the problem of providing a therapeutic agent for a disease in which formation of tertiary lymphoid tissue occurs in a lesion.　This prophylactic or therapeutic agent for a disease in which formation of tertiary lymphoid tissue occurs in a lesion contains an anti-CD153 antibody having cytotoxicity or an antigen-binding fragment of the antibody.

Description

Preventive or therapeutic drugs for diseases in which tertiary lymphatic tissue is formed in the affected area

[0001] The present disclosure relates to a preventive or therapeutic drug for a disease in which tertiary lymphoid tissue (TLT) is formed at the lesion site.

[0002] TLTs are induced ectopic lymphoid tissues, which resemble lymph nodes and are formed in non-lymphoid tissues (Non-Patent Documents 1-3). TLTs are infiltrated with large numbers of lymphocytes and serve as the origin of adaptive immune responses through the interaction and proliferation of T cells and B cells, amplifying the immune response. For example, in kidney diseases such as pyelonephritis, IgA nephropathy, and lupus nephritis, the induction of TLTs has been reported to cause prolonged inflammation and repair failure (Non-Patent Document 4). It has also been reported that the presence of highly mature TLTs in transplanted kidneys is associated with a higher risk of renal function decline five years later (Non-Patent Document 5).

[0003] Furthermore, it has been reported that the interaction between senescence-associated T cells (SAT) and age-associated B cells (ABC), which increase with age, is involved in the expansion and maturation of TLTs in the kidney and the aggravation of renal dysfunction (Non-Patent Document 6).

[0004] Sato Y. et al., Immunology of the aging kidney. Nat Rev Nephrol. 2019;15(10):625-40.Antonioli L. et al. Ectopic Lymphoid Organs and Immune-Mediated Diseases: Molecular Basis for Pharmacological Approaches. Trends Mol Med. 2020;26(11):1021-1033.Sautes-Fridman C. et al. Tertiary lymphoid structures in theera of cancer immunotherapy. Nat Rev Cancer. 2019;19(6):307-25.Sato Y. et al. Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney. JCI Insight. 2016;1(11):e87680.Lee H. et al., Advanced Tertiary Lymphoid Tissues in Protocol Biopsies are Associated with Progressive Graft Dysfunction in Kidney Transplant Recipients. J Am Soc Nephrol. 2022;33(1):186-200. doi: 10.1681 / ASN.2021050715. Yuki Sato et al. CD153 / CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury. J.Clin.Invest. 2022;132(2):e146071. https: / / doi.org / 10.1172 / JCI146071.

[0005] An object of the present disclosure is to provide a preventive or therapeutic agent for diseases in which TLT formation occurs in the lesions.

[0006] The present inventors have found that administration of an anti-CD153 antibody or an antigen-binding fragment of that antibody with antibody-dependent cell-mediated cytotoxicity in a mouse model of renal TLT formation reduces TLTs, improves fibrosis, and reduces the number of repair-impaired tubules in renal tissue. Based on these findings, the inventors have found that an anti-CD153 antibody or an antigen-binding fragment of that antibody with cytotoxic activity can be used as a preventive or therapeutic agent for diseases in which TLTs are formed in lesions. The present disclosure was completed based on these findings and through further investigation.

[0007] The present disclosure provides the following aspects of the invention: Item 1. A prophylactic or therapeutic drug for a disease in which TLT formation occurs in lesions, comprising an anti-CD153 antibody or an antigen-binding fragment of the antibody having cytotoxic activity. Item 2. The prophylactic or therapeutic drug of Item 1, wherein the anti-CD153 antibody is a monoclonal antibody. Item 3. The prophylactic or therapeutic drug of Item 1 or 2, wherein the anti-CD153 antibody is an IgG. Item 4. The prophylactic or therapeutic drug of any of Items 1 to 3, wherein the anti-CD153 antibody is a human antibody, a humanized antibody, or a chimeric antibody. Item 5. The prophylactic or therapeutic drug of any of Items 1 to 4, wherein the anti-CD153 antibody or the antigen-binding fragment of the antibody is an anti-CD153 antibody having antibody-dependent cell-mediated cytotoxicity (ADCC) activity, an anti-CD153 antibody having complement-dependent cytotoxicity (CDC) activity, an anti-CD153 antibody conjugated with a cytotoxic substance, or an antigen-binding fragment conjugated with a cytotoxic substance. Item 6. Item 7. The prophylactic or therapeutic agent according to any one of Items 1 to 5, used for the prevention or treatment of a disease in which TLT formation occurs in the kidney. Item 8. The prophylactic or therapeutic agent according to any one of Items 1 to 6, used for inhibiting the progression of kidney disease. Item 9. A method for the prevention or treatment of a disease in which TLT formation occurs in lesions, comprising administering an anti-CD153 antibody or an antigen-binding fragment of the antibody having cytotoxic activity to a patient suffering from a disease in which TLT formation occurs in lesions. Item 10. Use of an anti-CD153 antibody or an antigen-binding fragment of the antibody having cytotoxic activity for use in treatment for the prevention or treatment of a disease in which TLT formation occurs in lesions. Item 11. Use of an anti-CD153 antibody or an antigen-binding fragment of the antibody having cytotoxic activity for the manufacture of a prophylactic or therapeutic agent for a disease in which TLT formation occurs in lesions.

[0008] The preventive or therapeutic agent of the present disclosure can act specifically on TLT, thereby exerting effects such as suppressing TLT formation, reducing TLT, decreasing mature TLT, preventing the progression of TLT stages, and eliminating TLT.

[0009] Figure 1 shows a schematic diagram of the structure of TLTs formed in the kidney at each maturity stage. Figure 2 shows the protocol for an anti-CD153 antibody administration experiment using a mouse model of renal TLT formation. Figure 3 shows the results of the anti-CD153 antibody administration experiment, including images of damaged kidneys stained with periodic acid Schiff (PAS), the TLT area (%) calculated from the images, and the number of mature TLTs measured from damaged kidneys stained with fluorescent immunostaining. Figure 4 shows the results of the anti-CD153 antibody administration experiment, including flow cytometry analysis of cells obtained from the kidneys to identify CD153-positive senescence-associated T cells (CD45 + CD4 + CD44 high CD62L low PD1 + CD153 + Figure 5 shows the results of an experiment in which anti-CD153 antibodies were administered, and shows images of damaged kidneys stained with Picrosirius Red, along with the fibrosis area (%) calculated from the images. Figure 6 shows the results of an experiment in which anti-CD153 antibodies were administered, and shows images of damaged kidneys immunostained for Vcam1 and LTL, along with the percentage of renal tubules with impaired repair (%) calculated from the images.

[0010] 1. Terms and Definitions Unless otherwise specified, terms used herein have the meanings that are commonly understood by those skilled in the art of medicine, pharmacology, molecular biology, microbiology, organic chemistry, etc. If a term defined herein does not have the same meaning as commonly understood, the description in this specification shall take precedence.

[0011] In this disclosure, "tertiary lymphoid tissue (TLT)" refers to a lymph node-like tissue composed of lymphocyte aggregates, which is an inducible ectopic lymphoid tissue formed in non-lymphoid tissue. TLTs are classified into stages according to their degree of structural maturity. In this disclosure, the maturity of tertiary lymphoid tissue (TLT) is classified into stages I to III as follows, in accordance with the classification described in a literature article (Sato Y. et al. Kidney Int. 2020;98(2):448-463). For reference, Figure 1 shows a structural schematic diagram of TLTs formed in the kidney at each maturity stage. Stage 0 shown in Figure 1 represents unorganized lymphocytes, which are precursors of TLTs and do not correspond to the TLTs defined in this disclosure. Stage I: TLTs that do not contain follicular dendritic cells (FDCs) or germinal centers. Stage II: TLTs that contain FDCs but not germinal centers. Stage III: TLTs that contain FDCs and germinal centers.

[0012] In the present disclosure, an "antibody" refers to a molecule that binds to a specific antigen. Antibodies include heavy and light chains. The heavy chain includes a heavy chain variable region and a heavy chain constant region. The light chain includes a light chain variable region and a light chain constant region. The heavy and light chain variable regions each contain, from the N-terminus, CDR1, CDR2, and CDR3 as complementarity determining regions (CDRs) that recognize antigens. In the heavy and light chain variable regions, the CDRs are structurally supported by a region called the framework, except for CDR2. Antibodies may have two heavy chains and two light chains, or may be composed of one heavy chain and one light chain (sometimes referred to as a single-chain antibody). Antibodies are classified into isotypes, such as IgA, IgG, IgE, IgM, and IgY, which are further classified into subclasses, such as IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4.

[0013] In the present disclosure, "a preventive or therapeutic agent for a disease in which TLT formation occurs in the lesion" refers to an agent used to prevent or treat a disease in which TLT formation occurs in the lesion by suppressing TLT formation, reducing TLT, preventing TLT from progressing to a later stage, eliminating TLT, etc. In the present disclosure, "prevention" refers to suppressing the onset of a disease or lengthening the period until onset, and "treatment" refers to alleviating, mitigating, or slowing the rate of progression of a disease.

[0014] In the present disclosure, the term "antigen-binding fragment" of an antibody refers to an antibody fragment that retains the ability to bind to an antigen. Antigen-binding fragments of antibodies include a Fab fragment consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, and a CH1 domain that is part of the heavy chain variable region; a Fab' fragment consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, a CH1 domain that is part of the heavy chain variable region, and a hinge region; a F(ab')2 fragment in which two Fab' fragments are linked by a disulfide bridge at the hinge region; and an Fv fragment consisting of a heavy chain variable region and a light chain variable region.

[0015] In the present disclosure, "cytotoxic activity" refers to the activity of an antibody or antigen-binding fragment that binds to a cell, thereby inducing growth inhibition or cell death in the bound cell.

[0016] 2. Prophylactic or therapeutic agents for TLTs The present disclosure provides prophylactic or therapeutic agents for diseases in which TLTs are formed in lesions, comprising an anti-CD153 antibody or an antigen-binding fragment of the antibody having cytotoxic activity. The prophylactic or therapeutic agents of the present disclosure are described below.

[0017] [Active ingredient] The preventive or therapeutic agent of the present disclosure uses an anti-CD153 antibody or an antigen-binding fragment of the antibody with cytotoxic activity as an active ingredient. The expansion of TLTs is related to the binding of CD153 expressed on senescence-associated T cells (SAT cells) to CD30 expressed on senescence-associated B cells (Ag-associated B cells: ABC). Furthermore, it has been confirmed that antibodies without cytotoxic activity against SAT cells have no preventive or therapeutic effect. Therefore, the preventive or therapeutic agent of the present disclosure not only inhibits this binding with the anti-CD153 antibody or the antigen-binding fragment of the antibody, but also confers cytotoxic activity on SAT cells, thereby enabling the prevention or treatment of TLTs.

[0018] In the present disclosure, the anti-CD153 antibody may be any antibody that recognizes the extracellular region of CD153 on SAT cells as an epitope. The anti-CD153 antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. Furthermore, the anti-CD153 antibody may be of any isotype, such as IgG, IgA, IgE, IgM, or IgY, as long as it has cytotoxic activity, but is preferably IgG.

[0019] Furthermore, in the present disclosure, the anti-CD153 antibody may be any of a human antibody, a humanized antibody, and a chimeric antibody. A human antibody is an antibody derived from a human. A humanized antibody is an antibody in which a non-human CDR sequence is grafted onto the framework region of a human antibody, and is composed of a non-human antibody CDR, a human antibody framework region, and a human antibody constant region. A chimeric antibody is an antibody composed of a variable region of one species and a constant region of another species. Chimeric antibodies include antibodies composed of a variable region derived from a non-human source and a human constant region.

[0020] Examples of anti-CD153 antibodies with cytotoxic activity include anti-CD153 antibodies with antibody-dependent cell-mediated cytotoxicity (ADCC) activity, anti-CD153 antibodies with complement-dependent cytotoxicity (CDC) activity, and anti-CD153 antibodies conjugated to cytotoxic substances.

[0021] Anti-CD153 antibodies having ADCC or CDC activity can be produced by known techniques, and methods for confirming the presence or absence of ADCC or CDC activity in antibodies are also known.

[0022] An anti-CD153 antibody conjugated with a cytotoxic substance can be prepared by conjugating a cytotoxic substance to an anti-CD153 antibody. The cytotoxic substance may be any substance capable of inducing cell death in SAT cells, and examples thereof include toxins, radioactive substances, and chemotherapeutic agents. Toxins are proteins or polypeptides that exhibit cytotoxicity, such as diphtheria toxin. Radioactive substances are substances containing radioisotopes, such as 32 P, 14 C. 125 I, 3 H, 131 I, 186 Re, 188 Examples include substances containing radioactive isotopes such as Re. Chemotherapeutic agents are substances other than radioactive substances that have cytotoxic activity.

[0023] Among the anti-CD153 antibodies used in the present disclosure, preferred examples include anti-CD153 antibodies having ADCC activity or CDC activity, and particularly preferred examples include anti-CD153 antibodies having ADCC activity.

[0024] The antigen-binding fragment of the anti-CD153 antibody used in the present disclosure may be any fragment that binds to CD153 expressed on SAT cells and exhibits cytotoxic activity, and examples thereof include an antigen-binding fragment of the anti-CD153 antibody bound to a cytotoxic substance.

[0025] In the preventive or therapeutic agents of the present disclosure, one or more of the anti-CD153 antibodies having cytotoxic activity and antigen-binding fragments of the antibodies having cytotoxic activity may be used alone or in combination as the active ingredient.

[0026] [Formulation] The prophylactic or therapeutic agent of the present disclosure is formulated into a desired dosage form using at least one pharmaceutically acceptable carrier and provided as a pharmaceutical composition. The type of carrier used to formulate the prophylactic or therapeutic agent of the present disclosure is not particularly limited, as long as it is non-toxic to recipients at the dose and concentration used, and known carriers (Remington's Pharmaceutical Sciences, 18 th Ed. Mack Printing Company, 1990) depending on the dosage form. Pharmaceutically acceptable carriers include, but are not limited to, aqueous media such as sterile water and physiological saline; buffers such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, and benzyl alcohol; preservatives such as methylparaben, propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; polysorbate Examples of surfactants include surfactants such as sorbate 80, polysorbate 20, and poloxamer 188; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; sugars such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran; chelating agents such as EDTA and EGTA; low-molecular-weight (less than about 10 residues) oligopeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as polyethylene glycol.

[0027] The dosage form of the pharmaceutical composition comprising the prophylactic or therapeutic agent of the present disclosure may be appropriately determined depending on the administration method, etc., and may be any of solid, semi-solid, liquid, etc. One embodiment of the pharmaceutical composition comprising the prophylactic or therapeutic agent of the present disclosure is a lyophilized preparation. Another embodiment of the pharmaceutical composition comprising the prophylactic or therapeutic agent of the present disclosure is an aqueous solution.

[0028] The dosage form of the pharmaceutical composition containing the prophylactic or therapeutic agent of the present disclosure may be appropriately determined depending on the administration method, etc., and examples thereof include parenteral administration preparations such as injections, drip infusions, suppositories, injections, drip infusions, suppositories, aerosol inhalants, eye drops, lotions, gels, sprays, and ointments; and oral administration preparations such as capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewable preparations, buccal preparations, pastes, syrups, suspensions, elixirs, and emulsions. Among these, preferred examples include parenteral administration preparations.

[0029] [Application] Animals to which the preventive or therapeutic agent of the present disclosure is applicable are not particularly limited, and examples include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, etc. Among these, humans are preferred.

[0030] The preventive or therapeutic agent of the present disclosure is used for the purpose of inhibiting TLT formation in lesions, reducing TLT, preventing the progression of TLT stages, reducing mature TLT, eliminating TLT, etc. That is, the patient to whom the preventive or therapeutic agent of the present disclosure is administered is a patient suffering from a disease in which TLT formation occurs in lesions. When the preventive or therapeutic agent of the present disclosure is administered to the patient at a stage in which TLT has not yet been formed in the lesions, TLT formation in the lesions can be inhibited and the progression of the pathology can be suppressed. Furthermore, when the preventive or therapeutic agent of the present disclosure is administered to the patient at a stage in which TLT has already been formed in the lesions, it becomes possible to inhibit the progression of the pathology by reducing TLT in the lesions, reducing mature TLT, or inhibiting the progression of TLT stages.

[0031] Diseases in which TLT formation occurs in lesions include, but are not limited to, kidney disease (particularly chronic kidney disease). More specific examples of kidney disease include IgA nephropathy, lupus nephritis, ANCA-associated vasculitis, diabetic nephropathy, nephrosclerosis, pyelonephritis, chronic renal failure, and kidney transplantation. The preventive or therapeutic agent of the present disclosure can suppress intrarenal TLT formation, prevent TLT progression, and eliminate TLT in kidneys of kidney disease patients, thereby enabling the suppression of kidney disease progression.

[0032] [Dosage and Administration] The administration method of the prophylactic or therapeutic agent of the present disclosure is not particularly limited and may be appropriately determined depending on the type of active ingredient used, the type of disease to be treated, etc. Examples of administration methods include oral administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intraarticular administration, intralesional administration, intravesical administration, pulmonary administration, nasal administration, etc. Among these, intravenous administration and subcutaneous administration are preferred.

[0033] The dosage of the prophylactic or therapeutic agent of the present disclosure may be appropriately determined depending on the type of disease to be treated, the age and body weight of the patient, etc. For example, one example of the dosage of the prophylactic or therapeutic agent of the present disclosure is approximately 0.1 to 10 mg / kg of body weight of the anti-CD153 antibody or antigen-binding fragment thereof administered once daily to an adult. The administration interval of the prophylactic or therapeutic agent of the present disclosure may be appropriately determined within a range that achieves the preventive or therapeutic effect for PH, and may include, for example, daily, every other day, weekly, every two to three weeks, monthly, every other month, or every two to three months.

[0034] The present disclosure will be explained in more detail below by showing examples, but it should not be construed as being limited to these examples.

[0035] 1. Method 1-1. Preparation of anti-CD153 antibodies The mcRM153 antibody (an anti-mouse CD153 antibody with ADCC activity, a rat-mouse chimeric antibody) and the LALA antibody (an anti-mouse CD153 antibody without ADCC activity, a rat-mouse chimeric antibody) were prepared according to the following procedure.

[0036] (1) Obtaining the primary amino acid sequence of the Fv portion of the RM153 antibody. Sequence analysis of the Fv variable region of the anti-mouse CD153 rat antibody (clone name RM153) was performed with reference to the following literature (Tran NC, et al., Scientific Reports, 6:31730 | DOI: 10.1038 / srep31730). Specifically, first, the RM153 antibody (eBioscience TM The RM153 antibody (Catalog # 14-1531-85) was reductively alkylated with DTT (DL-dithiothreitol) according to standard methods and then adsorbed to magnetic beads. The glycans were cleaved using PNGase, and the sample was digested with various proteases. The purified sample was then analyzed by LC-MS (nanoLC-MS, Q Exactive) using a Stage Tip (SCX-SDB). The resulting MS / MS data was analyzed denovo using PEAKS software. The sequences of the Fv variable regions of the heavy and light chains of the RM153 antibody were identified by assembly based on the resulting peptide sequences.

[0037] (2) Construction of Rat-Mouse Chimeric Antibody Expression Plasmid Vectors. Based on the primary amino acid sequence information of the identified RM153 antibody's heavy chain variable region (VH) and light chain variable region (VL), cDNAs encoding each protein were designed using the appropriate genetic code. Using the obtained VH and VL sequences, expression plasmid vectors for the RM153 rat-mouse chimeric antibody (mcRM153) and a mutant antibody (LALA antibody) lacking antibody-dependent cellular cytotoxicity (ADCC) activity due to leucine-to-alanine mutations at positions 234 and 235 were constructed using the method described previously ( Molecular Immunology 63 (2015) 456-463. doi: 10.1016 / j.molimm.2014.09.017 ). Specifically, the heavy chain of a chimeric antibody whose variable region is the VH of the RM153 antibody and whose constant region is the constant region of mouse IgG2a (UniProt # P84751.1), or a heavy chain with LALA mutations (L234A / L235A) introduced into the same sequence, and the light chain of a chimeric antibody whose variable region is the VL of the RM153 antibody and whose constant region is the constant region of mouse kappa chain (UniProt # P01837.2) were cloned into the pcDNA3.1 plasmid vector (Invitrogen).

[0038] (3) Production and Purification of Rat-Mouse Chimeric Antibodies Rat-mouse chimeric antibodies were produced using the Expi293 Expression System (Life Technologies). Plasmids were transfected into Expi293F cells according to the manufacturer's instructions, and transient expression was achieved in the culture supernatant. The chimeric antibodies contained in the collected culture supernatant were purified by affinity purification using a Protein A column according to standard methods [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Goding, James W. ISBN 10: 0122870239, Academic Press]. The specific binding of the purified mcRM153 and LALA antibodies to the CD153 antigen was confirmed by flow cytometry using mouse CD153 antigen-expressing cells. The resulting mcRM153 and LALA antibodies bound specifically to the CD153 antigen with similar high affinity and inhibited CD153-CD30 binding.

[0039] (4) Evaluation of Antibody ADCC Activity Antibody ADCC activity was measured using the Mouse FcγRIV ADCC Bioassay (Promega). Specifically, the procedure was as follows: HEK293 cells stably expressing mouse CD153 were cultured overnight in a 96-well half-area plate and allowed to adhere. The mcRM153 or LALA antibody, which had been developed against these cells, was then added along with effector cells. The effector cells expressed mFcγRIV and contained a luciferase reporter downstream of NFAT (Nuclear factor of activated T-cells) to detect downstream intracellular signaling. After incubation in a 37°C, 5% CO2 incubator for 6 hours, a colorimetric reagent was added and incubated for 5 minutes. The fluorescent signal was then detected using a luminometer. The results demonstrated that the mcRM153 antibody exhibited high ADCC activity, while the LALA antibody completely abolished ADCC activity.

[0040] 1-2. Renal TLT Formation Mouse Model. Unilateral renal ischemia-reperfusion injury was induced in 12-month-old wild-type mice (C57BL6 / J). Specifically, under isoflurane inhalation anesthesia, a left flank incision was made to expose the kidney. The adipose tissue around the renal hilum was dissected, and the renal hilum was clamped with clips to occlude blood flow. After the desired time, the clips were released to allow reperfusion, and the incision was closed. The entire procedure, from incision to closure, was performed on a warmer to prevent hypothermia. In this mouse model, small tertiary lymphoid tissue formed at least 14 days after surgery, and mature tertiary lymphoid tissue, primarily composed of blood vessels, formed by 45 days after surgery.

[0041] 1-3. Anti-CD153 Antibody Administration Experiments: To the mouse model of renal tertiary lymphoid tissue formation described above, 150 μg of each of the mcRM153 antibody (anti-CD153 antibody group), the LALA antibody (LALA group), and the mouse IgG2a isotype control (control group) were intraperitoneally administered in 200 μL of sterile phosphate buffer every 7 days, starting from the day of unilateral renal ischemia-reperfusion injury (Figure 2). On postoperative day 45, general anesthesia was induced by intraperitoneal administration of 10 μL / BWg of a triple-anesthesia mixture of medetomidine (75 μg / mL), midazolam (400 μg / mL), and butorphanol (500 μg / mL). Whole blood and organs were then collected. The collected tissues were divided and subjected to RNA extraction, formalin-fixation, and flow cytometric analysis.

[0042] 1-4. Histological Analysis Histological analysis was performed using fixed paraffin sections cut at the center of the injured kidney. To assess TLT size, photographs of periodic acid Schiff (PAS) stained TLTs were taken, and the area of ​​the TLT and renal cortex was measured using Photoshop. The ratio was defined as the TLT area for that individual. Mature TLTs were counted by immunofluorescence staining, where the internal fibroblasts were positive for CD21 (a follicular dendritic cell marker). Renal fibrosis was assessed by photographing Picrosirius Red stained TLTs and measuring the positive area and the cross-sectional area of ​​the kidney using Image J. The ratio was defined as the fibrotic area. The proportion of incompetent tubules was determined by immunofluorescence staining for Vcam1, a marker for incompetent tubules, and LTL (Lotus tetragonolobus lectin), a marker for repaired proximal tubules. Seven fields of view from each kidney were photographed, and the number of positive tubules in each field was counted. The ratio of Vcam1-positive tubules to Vcam1- or LTL-positive tubules was used to determine the proportion of incompetent tubules.

[0043] 1-5. Flow Cytometry For flow cytometry, kidneys were manually minced with a scalpel and then treated with HBSS(-) (phenol red-free Hanks' Balanced Salt Solution) containing 1 mg / mL collagenase and 0.1 mg / mL DNase I at 37°C for 20 minutes, then passed through a cell strainer to obtain single cells. Spleens were bluntly isolated into single cells, hemolyzed, and used for analysis. The single-cell solution was subjected to Fc receptor blocking using FcR Blocking reagent (Miltenyi Biotec), then reacted with fluorescently labeled antibodies, and analyzed using a FACS Canto II (BD).

[0044] 2. Results: In the damaged kidneys of the control group, fully enlarged and mature TLTs were observed (Figure 3). In contrast, in the anti-CD153 antibody group, TLTs in the damaged kidneys were smaller and fewer mature TLTs were observed (Figure 3). Furthermore, flow cytometry analysis confirmed that CD153-positive senescence-associated T cells in the kidneys were reduced in the anti-CD153 antibody group (Figure 4). In addition, histological analysis confirmed that the anti-CD153 antibody group had fewer fibrotic areas and repair-impaired tubules, but these effects were not observed in the LALA group (Figures 5 and 6).

[0045] These results demonstrate that administration of cytotoxic anti-CD153 antibodies suppresses TLT formation after renal injury and alleviates renal injury. Furthermore, since non-cytotoxic anti-CD153 antibodies were unable to suppress TLT formation, it is important that anti-CD153 antibodies used to treat TLTs have cytotoxicity.

Claims

1. A preventive or therapeutic drug for diseases in which tertiary lymphoid tissue is formed at the lesion site, comprising an anti-CD153 antibody or an antigen-binding fragment of that antibody having cytotoxic activity.

2. The preventive or therapeutic drug according to claim 1, wherein the anti-CD153 antibody is a monoclonal antibody.

3. The preventive or therapeutic drug according to claim 1 or 2, wherein the anti-CD153 antibody is an IgG.

4. The preventive or therapeutic agent according to claim 1 or 2, wherein the anti-CD153 antibody is a human antibody, a humanized antibody, or a chimeric antibody.

5. The preventive or therapeutic agent according to claim 1 or 2, wherein the anti-CD153 antibody or antigen-binding fragment thereof is an anti-CD153 antibody having antibody-dependent cell-mediated cytotoxicity (ADCC) activity, an anti-CD153 antibody having complement-dependent cytotoxicity (CDC) activity, an anti-CD153 antibody conjugated with a cytotoxic substance, or an antigen-binding fragment conjugated with a cytotoxic substance.

6. The prophylactic or therapeutic agent according to claim 1 or 2, which is used for the prevention or treatment of a disease in which tertiary lymphoid tissue is formed in the kidney.

7. A preventive or therapeutic drug according to claim 1 or 2, which is used to inhibit the progression of kidney disease.

8. A method for preventing or treating a disease in which tertiary lymphoid tissue is formed in the lesion, comprising administering an anti-CD153 antibody or an antigen-binding fragment of that antibody having cytotoxic activity to a patient suffering from the disease in which tertiary lymphoid tissue is formed in the lesion.

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