Autoantibody adsorbent with controlled orientation

Abstract

Provided is a complex comprising an autoantigen protein or an antibody-binding fragment thereof specifically bound by a disease-specific autoantibody, and a substrate, the autoantigen protein or fragment being immobilized on the surface of the substrate with controlled orientation.

Description

Orientation-controlled autoantibody adsorbent

[0001] The present invention relates to an orientation-controllable adsorbent for adsorbing autoantibodies, which can be used in the treatment of autoimmune diseases and the like.

[0002] Autoimmune diseases are intractable diseases that occur when the immune system loses balance, causing the body to recognize and attack its own cells and tissues as antigens (autoantigens), and the disease progresses with the development of disease-specific autoantibodies. Conventionally, autoimmune diseases have been treated by suppressing the patient's immune function using immunosuppressants such as steroids and cyclosporine, and antibody production inhibitors such as anti-CD20 antibodies.

[0003] Furthermore, apheresis is also used to treat autoimmune diseases, in which blood is extracted from a patient and subjected to plasma exchange or plasma adsorption to remove antibodies and immune complexes from the patient's blood. Plasma adsorbents developed include carriers with tryptophan or phenylalanine immobilized as ligands (Imusova TR, Imusova PH (manufactured by Asahi Kasei Medical Co., Ltd.)) and carriers with dextran sulfate immobilized (Ceresorb (manufactured by Kaneka Medics Corporation)). Antibody adsorbents with certain peptides immobilized as ligands have also been reported (see, for example, Patent Document 1).

[0004] However, these therapies have safety concerns because they nonspecifically suppress the immune system, and therefore there is a need for novel therapies that are highly safe and have efficacy equal to or greater than that of conventional therapies.

[0005] Patent No. 6259245 specification

[0006] Artificial Organs 13(2): 997-1000, 1984, Yoshiya Yamazaki et al., Basic and Clinical Studies of Immunoadsorbents (IM-P) Journal of the Japanese Society of Apheresis 16, 327, 1997, Hidetoshi Kutsugi et al., Selective Adsorption Therapy Using Antigens, Antibodies, and Synthetic Ligands

[0007] Therefore, an object of the present invention is to develop a novel autoantibody adsorbent that is highly safe and has sufficient therapeutic effect compared to conventional methods.

[0008] As a result of intensive research to solve the above problems, the inventors succeeded in developing a carrier that can efficiently adsorb autoantibodies by immobilizing autoantigen proteins, which are recognized by disease-specific autoantibodies, as ligands on the carrier with controlled orientation.

[0009] That is, the present invention provides the following aspects: (1) A complex comprising an autoantigen protein or an antibody-binding fragment thereof that is specifically bound by a disease-specific autoantibody, and a substrate, wherein the autoantigen protein or fragment is immobilized on the surface of the substrate with controlled orientation. (2) The complex according to (1), wherein the autoantigen protein is selected from the group consisting of desmoglein 3, desmoglein 1, muscle-specific receptor tyrosine kinase, nicotinic acetylcholine receptor, glomerular basement membrane type 4 collagen, BP180, BP230, aquaporin 4, myelin oligodendrocyte glycoprotein, NMDA receptor, membrane-type phospholipase A2 receptor, and thrombospondin type 1 domain-containing 7A. (3) The complex according to (1) or (2), wherein the autoantigen protein or fragment is immobilized on the surface of the substrate with controlled orientation via an avidin-biotin bond, an ionic bond, a chelate bond, a covalent bond, or an antigen-antibody bond. (4) The conjugate according to (3), wherein the avidin is deglycosylated avidin. (5) The conjugate according to any one of (1) to (4), wherein the substrate is a solid carrier or support. (6) A disease-specific autoantibody adsorbent comprising the conjugate according to any one of (1) to (5). (7) The disease-specific autoantibody adsorbent according to (6), for use in the treatment or prevention of a disease selected from the group consisting of pemphigus, myasthenia gravis, anti-glomerular basement membrane nephritis, pemphigoid, neuromyelitis optica, anti-NMDA receptor encephalitis, and membranous nephropathy. (8) A column comprising the disease-specific autoantibody adsorbent according to (6). (9) The column according to (8), which is used for apheresis. (10) A method for treating an autoimmune disease, comprising using the disease-specific autoantibody adsorbent according to (6) or (7). (11) A method for treating an autoimmune disease, comprising using the disease-specific autoantibody adsorbent according to (8) or (9).

[0010] According to the present invention, an autoantigen protein or its antibody-binding fragment, which is specifically recognized and bound by a disease-specific autoantibody that causes an autoimmune disease, is immobilized on the surface of a substrate with controlled orientation, and the resulting orientation-controlled autoantibody adsorbent can efficiently adsorb the target autoantibody. Furthermore, since the autoantigen specific to the target disease is immobilized on the substrate, the autoantibody adsorbent obtained by the present invention specifically adsorbs the autoantibody specific to the target disease. Therefore, use of the orientation-controlled autoantibody adsorbent of the present invention can provide an autoimmune disease treatment that is highly specific to the treatment target and highly efficient at removing pathogenic autoantibodies.

[0011] 1. Target Diseases The target disease of the present application may be any disease in which the appearance of autoantibodies causes the onset and / or progression of the disease, and is not particularly limited. Autoantibodies are known to be involved in the onset and progression of various autoimmune diseases. Examples include, but are not limited to, pemphigus, myasthenia gravis, anti-glomerular basement membrane nephritis, pemphigoid, neuromyelitis optica, anti-NMDA (N-methyl-D-aspartate) receptor encephalitis, and membranous nephropathy.

[0012] 2. Disease-Specific Autoantibodies As used herein, the term "autoantibody" refers to an antibody produced within the body that attacks one's own cells or tissues, and is an antibody involved in the onset and / or progression of an autoimmune disease. Disease-specific autoantibodies are produced in a disease-specific manner in patients suffering from an autoimmune disease. Such autoantibodies are absent or almost absent in healthy individuals or patients suffering from diseases other than the autoimmune disease (including other autoimmune diseases).

[0013] Examples of disease-specific autoantibodies include, but are not limited to, anti-desmoglein 3 (DSG3) antibody, anti-desmoglein 1 (DSG1) antibody, etc., as examples of pemphigus-specific autoantibodies; anti-muscle-specific receptor tyrosine kinase (MuSK) antibody, anti-nicotinic acetylcholine receptor (AChR) antibody, etc., as examples of myasthenia gravis-specific autoantibodies; anti-glomerular basement membrane (GBM) antibody, as examples of anti-glomerular basement membrane nephritis-specific autoantibodies; and anti-pemphigoid-specific autoantibodies. Examples of autoantibodies specific to neuromyelitis optica include anti-aquaporin 4 (AQP4) antibody and anti-myelin oligodendrocyte glycoprotein (MOG) antibody, etc. Examples of autoantibodies specific to anti-NMDA receptor encephalitis include anti-NMDA receptor (NMDAR) antibody, etc. Examples of autoantibodies specific to membranous nephropathy include anti-membrane phospholipase A2 receptor (PLA2R) antibody and anti-thrombospondin type 1 domain-containing 7A (THSD7A) antibody, etc.

[0014] 3. Autoantigen Protein or Antibody-Binding Fragment As used herein, the term "autoantigen" refers to an antigen that is the target of an autoantibody and is specifically recognized and bound by the autoantibody. As used herein, the term "antibody-binding fragment" of an autoantigen protein refers to a fragment of the autoantigen protein that is specifically recognized and bound by the autoantibody in the same way as the full-length autoantigen protein, i.e., a fragment that retains the ability to bind to a specific antibody, or a fragment that includes a region capable of binding to an autoantibody. For example, when the autoantigen protein is a transmembrane protein, a fragment including an extracellular region can be used as a fragment of the autoantigen protein.

[0015] Examples of autoantigen proteins include, but are not limited to, examples of autoantigens specific to pemphigus such as desmoglein 3 (DSG3) and desmoglein 1 (DSG1); examples of autoantigens specific to myasthenia gravis such as muscle-specific receptor tyrosine kinase (MuSK) and nicotinic acetylcholine receptor (AChR); examples of autoantigens specific to anti-glomerular basement membrane nephritis such as type 4 collagen of the glomerular basement membrane (GBM); and examples of autoantigens specific to pemphigoid such as hemides of the epidermal basement membrane. Examples of autoantigens specific to neuromyelitis optica include aquaporin 4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG), examples of autoantigens specific to anti-NMDA receptor encephalitis include NMDA receptor (NMDAR), and examples of autoantigens specific to membranous nephropathy include membrane-type phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A (THSD7A).

[0016] The origin of the autoantigen protein or antibody-binding fragment (hereinafter simply referred to as "autoantigen protein or fragment") used in the present application is not particularly limited, but is preferably the same species as the animal suffering from or likely to suffer from the target disease, i.e., the same species as the source of the autoantibody to be adsorbed. The amino acid sequence information of the autoantigen protein may be obtained from a publicly known database (e.g., GenBank). Furthermore, the autoantigen protein or fragment is not limited to native forms containing mature or immature amino acid sequences, and may be any mutant in which one or more amino acids have been added, inserted, substituted, and / or deleted, as long as it retains specific binding to the target autoantibody. Such mutants include, but are not limited to, mutants containing amino acid sequences that have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence of the wild-type protein. The sequence identity of an amino acid sequence can be determined using methods well known to those skilled in the art, sequence analysis software, or the like. Examples of sequence analysis software include the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm.

[0017] For example, the autoantigen protein or fragment may have another peptide attached thereto (e.g., attached to the C-terminus), or the autoantigen protein or fragment may have another peptide attached thereto (e.g., attached to the C-terminus) and biotin attached thereto. Examples of other peptides include, but are not limited to, coiled-coil sequences (e.g., acidic tail sequences or basic tail sequences), tag (or label) sequences, etc. These other peptides are used in the production of autoantigen proteins or fragments for purposes such as facilitating multimerization (e.g., dimerization) (when the autoantigen protein has a multimeric form), purification, or immobilization on a support. The other peptide may be attached via a linker sequence. The linker sequence can be appropriately selected by those skilled in the art, and may be, for example, a peptide linker consisting of about 1 to 10 amino acids such as GGGGSGGGGS, or about 2 to 5 amino acids such as GGGGS, including glycine or serine.

[0018] 4. Substrate The substrate used in the present application is an insoluble solid carrier or support, and includes those in various shapes, such as, but not limited to, spheres (e.g., particles, beads, etc.), plates, sheets, fibers, membranes, hollow fiber membranes, gels, films, and combinations thereof.

[0019] Examples of substrate materials include, but are not limited to, carbohydrate materials such as agarose, dextran, cellulose, and activated carbon, synthetic polymers such as polymethacrylate, polystyrene, polyacrylamide, polyamide, polysulfone, polyethylene, polyvinyl alcohol, polypropylene, polyacrylonitrile, polyethylene phthalate, polyester, styrene-divinylbenzene copolymer, and ethylene-vinyl alcohol copolymer, inorganic materials such as silica gel, glass, and metal, and composite materials with metal added. Commercially available solid supports may also be used.

[0020] 5. Complex Comprising an Autoantigen Protein or Antibody-Binding Fragment and a Substrate According to the present invention, a complex (hereinafter also referred to as "the complex of the present application") can be obtained in which an autoantigen protein or fragment is immobilized on the surface of a substrate with controlled orientation. The method for immobilizing the autoantigen protein or fragment on the surface of the substrate may be any method that allows the autoantigen protein or fragment to be immobilized with controlled orientation, and examples of such methods include ionic bonding, covalent bonding, chelate bonding, antigen-antibody bonding, and avidin-biotin bonding, which are known in the art.

[0021] An example of a chelate binding method is a method that utilizes binding between a histidine tag added to an autoantigen protein or fragment and a divalent metal ion (e.g., Ni, Cu, Zn, Ca, Co, Fe, or Mg) immobilized in a chelated state on a substrate.

[0022] Antigen-antibody binding methods include, for example, methods using tags and anti-tag antibodies. For example, the binding between a tag attached to an autoantigen protein or fragment and an antibody specific to the tag immobilized on a substrate can be utilized. Such tags include, but are not limited to, peptide tags (e.g., Myc tags, FLAG tags, His tags, etc.), polypeptide tags, etc. Preferably, a monoclonal antibody is used as the anti-tag antibody. Commercially available products may be used as the substrate on which the divalent metal is immobilized and the substrate on which the anti-tag antibody is immobilized.

[0023] In the avidin-biotin binding method, in addition to avidin, avidin-like substances and modified avidins such as streptavidin, deglycosylated avidin (NeutrAvidin (registered trademark)), reversible avidin (Switchavidin), monomeric avidin (SAvPhire (registered trademark) monomeric streptavidin), avidin-like protein derived from mushrooms (Tamavidin (registered trademark) 2-REV), genetically modified streptavidin, or genetically modified avidin can also be used. The term "avidin" used herein includes not only avidin, but also avidin-like substances and modified avidins such as streptavidin, deglycosylated avidin (NeutrAvidin (registered trademark)), reversible avidin (Switchavidin), monomeric avidin (SAvPhire (registered trademark) monomeric streptavidin), avidin-like protein derived from mushrooms (Tamavidin (registered trademark) 2-REV), genetically modified streptavidin, and genetically modified avidin.

[0024] To produce the conjugate of the present application, for example, a substrate on which avidin has been immobilized and an autoantigen protein or fragment to which biotin has been attached may be used, thereby immobilizing the autoantigen protein or fragment on the substrate via the avidin-biotin bond. Commercially available substrates with avidin immobilized on their surfaces may also be used. Biotin may be added, for example, by attaching the sequence GLNDIFEAQKIEWHE (SEQ ID NO: 1) to the autoantigen protein or fragment, and then biotinylating only the K using biotin ligase. The peptide consisting of the sequence shown in SEQ ID NO: 1 is also known as Avi Tag (trademark).

[0025] The above-mentioned tag or biotin used to immobilize an autoantigen protein or fragment on a substrate surface is preferably added to a position away from the antibody-binding region of the autoantigen protein or fragment, preferably to the N-terminus or C-terminus away from the antibody-binding region. This ensures that, in the resulting complex of the present application, the antibody-binding regions of all (theoretically all) of the autoantigen proteins or fragments contained in the complex are located away from the substrate and are easily accessible. In this manner, the immobilization of all (theoretically all) of one or more autoantigen proteins or fragments on the substrate surface in a similar orientation such that their antibody-binding regions are located away from the substrate, i.e., at positions distal to the autoantibody-binding site, is referred to herein as "immobilized with controlled orientation."

[0026] The complex of the present invention thus obtained is an orientation-controllable autoantibody adsorbent that can efficiently adsorb disease-specific autoantibodies depending on the type of autoantigen protein or fragment contained therein.

[0027] 6. Treatment or Treatment Method for Autoimmune Disease The conjugate of the present application may be packed in a column or a bag such as a blood bag. Such a column or bag is preferably used for the treatment or prevention of a disease.

[0028] Therefore, a method for treating or preventing an autoimmune disease is provided, which is characterized by using the conjugate of the present application. When contacted with a sample, the conjugate of the present application can specifically remove autoantibodies contained in the sample, i.e., autoantibodies or autoantibody-producing B cells that recognize and bind to an autoantigen protein or fragment contained in the conjugate. The sample may be a biological sample obtained from an animal to be treated or prevented, such as blood (e.g., whole blood, serum, or plasma, preferably plasma). For example, autoantibodies or autoantibody-producing B cells in the blood may be removed by adsorbing them onto a column packed with the conjugate of the present application. The blood from which autoantibodies or autoantibody-producing B cells have been removed may be returned to the subject animal.

[0029] Therefore, an example of a treatment method is apheresis therapy for treating or preventing an autoimmune disease, which involves taking blood from an animal to be treated or prevented, treating the blood with the column, and then returning the blood to the animal.

[0030] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0031] Example 1: Antibody adsorption experiment using an anti-DSG3 antibody adsorption column Anti-DSG3 antibodies are known to be autoantibodies involved in the onset and progression of pemphigus. The extracellular domain of human desmoglein 3 (DSG3) was used as the autoantigen fragment. The amino acid sequence information for DSG3 is as follows: NCBI (National Center for Biotechnology Information) Accession No. NP_001935.2 Full length: 999 amino acids Signal peptide: amino acid numbers 1-23 Mature peptide: amino acid numbers 50-999 Cadherin repeat: amino acid numbers 82-155, 165-263, 272-375, 402-491 Transmembrane domain: amino acid numbers 616-640 Extracellular domain: amino acid numbers 50-615 (autoantibody-binding domain)

[0032] The extracellular domain of human desmoglein 3 (Kactus Biosystems, DSG-HM103) with a His tag attached to the C-terminus was immobilized on anti-His tag antibody agarose (Medical and Biological Laboratories, PM032-08) via the His tag to prepare a desmoglein 3 orientation-controlled adsorbent (DSG3 orientation-controlled adsorbent).

[0033] The desmoglein 3 protein was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via the amino group to prepare a desmoglein 3 control adsorbent (DSG3 control adsorbent).

[0034] Anti-desmoglein 3 antibody (Medical and Biological Laboratories, 7885) was added to the solution containing each of the above-mentioned adsorbents, and the mixture was mixed by inversion at 4°C for 12 hours, after which each supernatant was collected.

[0035] The amount of anti-desmoglein 3 antibody contained in the recovered supernatant was measured by ELISA. A microtiter plate was coated with 1 μg / mL desmoglein 3 overnight at 4°C, followed by washing and blocking. After washing, the supernatant was added and incubated at room temperature for 60 minutes. After washing, an HRP-labeled anti-human IgG antibody (Abcam, ab6759) was added and incubated at room temperature for 60 minutes. After washing, the chromogenic substrate 3,3',5,5'-tetramethylbenzidine was added, and the absorbance density (OD value) at 450 nm was measured to detect the anti-desmoglein 3 antibody. As a result, the amount of anti-desmoglein 3 antibody adsorbed by the DSG3 orientation-controlled adsorbent was 1.7 times that of the DSG3 control adsorbent (Table 1).

[0036] Example 2: Antibody adsorption experiment using an anti-DSG1 antibody adsorption column Anti-DSG1 antibodies are known to be autoantibodies involved in the onset and progression of pemphigus. The extracellular domain of human desmoglein 1 (DSG1) was used as the autoantigen fragment. The amino acid sequence information for DSG1 is as follows: NCBI Accession No. NP_001933 Full length: 1049 amino acids Signal peptide: amino acid numbers 1-23 Mature peptide: amino acid numbers 50-1049 Cadherin repeat: amino acid numbers 54-148, 161-265, 273-379, 397-484 Transmembrane domain: amino acid numbers 549-569 Extracellular domain: amino acid numbers 50-548 (autoantibody-binding domain)

[0037] A protein containing a His tag, the extracellular domain of human desmoglein 1, and a Myc tag (LifeSpan Biosciences Inc., LS-G56051) was immobilized to Anti-Myc-tag mAb-Magnetic Agarose (Medical and Biological Laboratories, M047-10) via the Myc tag to prepare a desmoglein 1 orientation-controlled adsorbent (DSG1 orientation-controlled adsorbent).

[0038] The desmoglein 1 protein was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via the amino group to prepare a desmoglein 1 control adsorbent (DSG1 control adsorbent).

[0039] Anti-desmoglein 1 antibody (Medical and Biological Laboratories, 7880) was added to the solution containing each of the above-mentioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4° C. The next day, the adsorbent was precipitated by centrifugation and washed with buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and then the anti-desmoglein 1 antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0040] The amount of anti-desmoglein 1 antibody in the collected solution was measured using an anti-desmoglein 1 antibody measurement kit (Medical and Biological Laboratories, 7880), and the antibody adsorption rate for each adsorbent was calculated, with the amount of antibody added being 100%. As a result, the anti-desmoglein 1 antibody adsorption rate for the DSG1 orientation-controlled adsorbent was 2.1 times that of the DSG1 control adsorbent (Table 1).

[0041] Example 3: Antibody adsorption experiment using an anti-MuSK antibody adsorption column Anti-MuSK antibodies are known to be autoantibodies involved in the onset and progression of myasthenia gravis. The extracellular domain of human muscle skeletal receptor tyrosine protein kinase (MuSK) was used as the autoantigen fragment. The amino acid sequence information for MuSK is as follows: NCBI Accession No. NP_005583.1 Full length: 869 amino acids Signal peptide: amino acid numbers 1-23 Mature peptide: amino acid numbers 24-869 Transmembrane domain: amino acid numbers 496-516 Extracellular domain: amino acid numbers 24-495 (autoantibody-binding domain)

[0042] The extracellular domain of human muscle-specific receptor tyrosine kinase (MuSK) (R&D Systems, AVI10189-050), which had an Avi tag and a His tag (6xHis) attached to its C-terminus and the Avi tag biotinylated, was immobilized on avidin agarose (Thermo Fisher Scientific, 29200) via avidin-biotin binding to prepare a MuSK orientation-controlled adsorbent.

[0043] The MuSK protein was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via the amino group to prepare a MuSK control adsorbent.

[0044] Anti-MuSK antibody (IBL International GmbH, RE51021) was added to the solution containing each of the above-mentioned adsorbents, and the mixture was mixed by inversion at 4°C for 12 hours, after which each supernatant was collected.

[0045] The amount of anti-MuSK antibody contained in the recovered supernatant was measured using an anti-MuSK antibody measurement kit (IBL International GmbH, RE51021). As a result, the amount of anti-MuSK antibody adsorbed by the MuSK orientation-controlled adsorbent was 1.8 times that of the MuSK control adsorbent (Table 1).

[0046] Example 4: Antibody adsorption experiment using anti-AChR antibody adsorption column Anti-nicotinic AChR antibodies (hereinafter referred to as "anti-AChR antibodies") are known to be autoantibodies involved in the onset and progression of myasthenia gravis. The nicotinic acetylcholine receptor (AChR) is a heteropentameric protein composed of two α chains, a β, a δ, and a γ or ε chain, and the main epitope site recognized by autoantibodies is known to be the α chain. Therefore, the extracellular domain of the α chain of human nicotinic AChR was used as the autoantigenic fragment. The amino acid sequence information constituting the α chain of AChR is as follows: NCBI Accession No. NP_001034612 Full length: 482 amino acids Signal peptide: amino acid numbers 1-20 Mature peptide: amino acid numbers 21-482 Transmembrane domain: amino acid numbers 263-471 Extracellular domain: amino acid numbers 22-262 (autoantibody-binding domain)

[0047] The α-chain extracellular domain (amino acid numbers 23-254) of the human nicotinic acetylcholine receptor (LifeSpan Biosciences, LS-G140545-50) with a His tag attached to the C-terminus was immobilized on nickel agarose (Thermo Fisher Scientific, 88831) via the His tag to prepare an acetylcholine receptor orientation-controlled adsorbent (AChR orientation-controlled adsorbent).

[0048] The acetylcholine receptor was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via an amino group to prepare an acetylcholine receptor covalently bonded adsorbent (AChR control adsorbent).

[0049] An anti-acetylcholine receptor antibody (EUROIMMUN, EA 1435-9601 G) was added to a solution containing each of the above-mentioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4°C. The next day, the adsorbent was precipitated by centrifugation and washed with buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and then the anti-acetylcholine receptor antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0050] The amount of anti-acetylcholine receptor antibody in the recovered solution was measured using an anti-acetylcholine receptor antibody measurement kit (EUROIMMUN, EA 1435-9601 G), and the antibody adsorption rate of each adsorbent was calculated, with the amount of antibody added being 100%. As a result, the anti-acetylcholine receptor antibody adsorption rate of the AChR orientation-controlling adsorbent was 1.6 times that of the AChR control adsorbent (Table 1).

[0051] Example 5. Antibody adsorption experiment using an anti-GBM antibody adsorption column Antibodies against type IV collagen in the glomerular basement membrane (anti-GBM antibodies) are known to be autoantibodies involved in the onset and progression of anti-glomerular basement membrane nephritis. Glomerular basement membrane type IV collagen is a trimer consisting of three α chains, and the NC1 domain present at the C-terminus of the α chain is known to be the autoantibody binding region. Therefore, the α chain NC region of human type IV collagen was used as the autoantigen fragment. The amino acid sequence information for type IV collagen is as follows: NCBI Accession No. NP_001836.3 UniprotKB P02462 Total length: 1669 amino acids Signal peptide: amino acid numbers 1-27 Mature peptide: amino acid numbers 173-1669 NC1 domain: amino acid numbers 1445-1669

[0052] The α-chain NC1 region (amino acid numbers 1444-1669) of human type IV collagen (LifeSpan Biosciences, LS-G11688-100) with a His tag attached to the N-terminus was immobilized on nickel agarose (Thermo Fisher Scientific, Pierce, 88831) via the His tag to prepare a type IV collagen orientation-controlled adsorbent (NC1 orientation-controlled adsorbent).

[0053] The above type IV collagen was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via an amino group to prepare a type IV collagen control adsorbent (NC1 control adsorbent).

[0054] Anti-GBM antibody (Thermo Fisher Diagnostics, Class II Immunoassay Series Area Anti-GBM Antibody) was added to the solution containing each of the aforementioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4°C. The next day, the adsorbent was precipitated by centrifugation and washed with buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and then the anti-GBM antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0055] The amount of anti-GBM antibody contained in the recovered solution was measured using an anti-GBM antibody measurement kit (Thermo Fisher Diagnostics, Class II Immunoassay Series Area Anti-GBM Antibody), and the antibody adsorption rate of each adsorbent was calculated, with the amount of antibody added being 100%. As a result, the anti-GBM antibody adsorption rate of the NC1 orientation-controlled adsorbent was 1.9 times that of the NC1 control adsorbent (Table 1).

[0056] Example 6: Antibody adsorption experiment using an anti-BP180 antibody adsorption column Antibodies (anti-BP180 antibodies) against BP180 (type XVII collagen), a component protein of hemidesmosomes present in the epidermal basement membrane, are known to be autoantibodies involved in the onset and progression of pemphigoid. BP180 is a homotrimer consisting of three identical α chains, and antibodies against an epitope present in the NC (non-collagenous) 16A region near the cell membrane of basal cells are known to be pathogenic. Therefore, the NC16A domain of human BP180 was used as the autoantigenic fragment. The amino acid sequence information for BP180 (type XVII collagen α1 chain) is as follows: NCBI Accession No. NP_000485.3 UniprotKB Q9UMD9 Full length 1497 amino acids NC16 domain: amino acids 1-566 NC16a domain: amino acids 488-566 (autoantibody binding region) Transmembrane domain: amino acids 468-488 Extracellular domain: amino acids 489-1497

[0057] A BP180 expression vector was constructed by inserting the BP180NC16A sequence, N-terminally linked to an Avi tag and a His tag, into the pcDNA3.4 TOPO vector (Invitrogen, A14697). The constructed expression vector was transfected into Chinese Hamster Ovary cells (CHO cells), and the protein expressed in the culture supernatant was purified using a nickel chelate column. The Avi tag site of the purified protein was biotinylated using biotin ligase (Avidity, BIRA) to produce biotinylated BP180. The biotinylated BP180 was immobilized on avidin agarose (Thermo Fisher Scientific, 29200) via avidin-biotin binding to prepare a BP180 orientation-controlled adsorbent.

[0058] The BP180 protein was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via the amino group to prepare a BP180 control adsorbent.

[0059] Anti-BP180 antibody (Medical and Biological Laboratories, 7695) was added to the solution containing each of the aforementioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4°C. The next day, the adsorbent was precipitated by centrifugation and washed with buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and then the anti-BP180 antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0060] The amount of anti-BP180 antibody contained in the recovered solution was measured using an anti-BP180 antibody measurement kit (Medical and Biological Laboratories, 7695), and the antibody adsorption rate of each adsorbent was calculated, assuming the amount of antibody added as 100%. As a result, the anti-BP180 antibody adsorption rate of the BP180 orientation-controlled adsorbent was 1.9 times that of the BP180 control adsorbent (Table 1).

[0061] Example 7. Antibody adsorption experiment using anti-AQP4 antibody adsorption column Anti-AQP4 antibodies are known to be autoantibodies involved in the onset and progression of neuromyelitis optica. The extracellular domain of human aquaporin 4 (AQP4) was used as the autoantigen fragment. The amino acid sequence information for AQP4 is as follows: NCBI Accession No. NP_001641 UniprotKB P55087 Total length: 323 amino acids Extracellular domain: amino acid numbers 216-315 (autoantibody binding region) Extracellular loop: 58-69, 137-155, 206-208, 223-231

[0062] Human aquaporin 4 (amino acid numbers 216-315) (LifeSpan Biosciences, LS-G141043-1) with a His tag attached to the N-terminus was immobilized on nickel agarose (Thermo Fisher Scientific, Pierce, 88831) via the His tag to prepare an aquaporin 4 orientation-controlled adsorbent (AQP4 orientation-controlled adsorbent).

[0063] The above-mentioned aquaporin 4 was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via an amino group to prepare an aquaporin 4 control adsorbent (AQP4 control adsorbent).

[0064] An anti-aquaporin 4 antibody (Cosmic Corporation, F0013E41) was added to a solution containing each of the aforementioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4°C. The next day, the adsorbent was precipitated by centrifugation and washed with a buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and the anti-aquaporin 4 antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0065] The amount of anti-aquaporin 4 antibody in the collected solution was measured using an anti-aquaporin 4 antibody assay kit (Cosmic Corporation, F0013E41), and the antibody adsorption rate for each adsorbent was calculated, with the amount of antibody added being 100%. As a result, the anti-aquaporin 4 antibody adsorption rate for the AQP4 orientation-controlled adsorbent was 1.6-fold higher than that for the AQP4 control adsorbent (Table 1).

[0066] Example 8. Antibody adsorption experiment using an anti-MOG antibody adsorption column Anti-MOG antibodies are known to be autoantibodies involved in the onset and progression of neuromyelitis optica. A fragment of human myelin oligodendrocyte glycoprotein (MOG) was used as the autoantigen fragment. The amino acid sequence information for MOG is as follows: NCBI Accession No. NP_002424.3 UniprotKB Q5SSB8 Full length 252 amino acids Signal peptide: amino acid numbers 1-29 Mature peptide: amino acid numbers 30-252

[0067] Myelin oligodendrocyte glycoprotein (amino acid numbers 30-154) (Bon Opus Biosciences LLC, BP169) with a His tag attached to the C-terminus was immobilized on nickel agarose (Thermo Fisher Scientific, Pierce, 88831) via the His tag to prepare a myelin oligodendrocyte glycoprotein orientation-controlled adsorbent (MOG orientation-controlled adsorbent).

[0068] The above myelin oligodendrocyte glycoprotein was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via an amino group to prepare a myelin oligodendrocyte glycoprotein control adsorbent (MOG control adsorbent).

[0069] Anti-MOG antibody (abcam, ab278115) was added to the solution containing each of the aforementioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4°C. The next day, the adsorbent was precipitated by centrifugation and washed with buffer (50 mM Tris, 150 mM NaCl, pH 7.2). The anti-MOG antibody bound to the adsorbent was then recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0070] The amount of anti-MOG antibody in the collected solution was measured using an anti-MOG antibody assay kit (abcam, ab278115), and the antibody adsorption rate for each adsorbent was calculated, with the amount of antibody added being 100%. As a result, the anti-MOG antibody adsorption rate for the MOG orientation-controlled adsorbent was 1.7 times that of the MOG control adsorbent (Table 1).

[0071] Example 9: Antibody adsorption experiment using an anti-NMDA receptor antibody adsorption column Anti-NMDA receptor antibodies are known to be autoantibodies involved in the onset and progression of anti-NMDA receptor encephalitis. NMDA receptors are heterotetramers consisting of GluN1 / GluN2 / GluN3 / GluN4 subunits, and it is known that autoantibodies bind primarily to the aminoterminal domain (ATD) composed of the N-terminal region of GluN1 or GluN2.As an autoantigenic fragment of NMDAR, a sequence in which the N-terminal domains of GluN1 and GluN2B are linked via a linker (GluN1: amino acid numbers 1-400; MSTMRLLTLALLFSCSVARAACDPKIVNIGAVLSTRKHEQMFREAVNQANKRHGSWKIQLNATSVTHKPNAIQMALSVCEDLISSQVYAILVSHPPTPNDHFTPTPVSYTAGFYRIPVLGLTTRMSIYSDKS) was used. IHLSFLRTVPPYSHQSSVWFEMMRVYSWNHIILLVSDDHEGRAAQKRLETLLEERESKAEKVLQFDPGTKNVTALLMEAKELEARVIILSASEDDAATVYRAAAMLNMTGSGYVWLVGERE ISGNALRYAPDGILGLQLINGKNESAHISDAVGVVAQAVHELLEKENITDPPRGCVGNTNIWKTGPLFKRVLMSSKYADGVTGRVEFNEDGDRKFANYSIMNLQNRKLVQVGIYNGTHVIP NDRKIIWPGGETEKPRGYQMSTRLKI (SEQ ID NO: 2), linker; GSTGGGGSGGGGSGGGGSGAASR (SEQ ID NO: 3), GluN2B: amino acids 29-408; QKSPPSIGIAVILVGTSDEVAIKDAHEKDDFHHLSVVPRVELVAMNETDPKSIITRICDLMSDRKIQGVVFADDTDQEAIAQILDFISAQTLTPILGIHGGSSMIMADKDESSMFFQFGPSIEQQASVMLNIMEEYDWYIFSIVTTYFPGY QDFVNKIRSTIENSFVGWELEEVLLLDMSLDDGDSKIQNQLKKLQSPIILLYCTKEEATYIFEVANSVGLTGYGYTWIVPSLVAGDTDTVPAEFPTGLISVSYDEWDYGLPARVRDGIAIITTAASDMLSEHSFIPEPKSSCYNTHEKRIYQSNMLNRYLINVTFEGRNLSFSEDGYQMHPKLVIILLNKERKWERVGKWKDKSLQMKYYVWPRMCPETEEQEDDHLSI (SEQ ID NO: 4)) was used.The amino acid sequence information of each subunit was obtained from the following: GluN1: NCBI Accession No. NP_007327 UniprotKB Q05586 GluN2B: NCBI Accession No. NP_000834 UniprotKB Q13224.

[0072] An NMDAR (ATD) expression vector was prepared by inserting a sequence in which a GGGGS linker, an Avi tag, and a His tag were linked to the C-terminal side of the autoantigen fragment into a pcDNA3.4 TOPO vector (Invitrogen, A14697).

[0073] The NMDA receptor protein was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via the amino group to prepare an NMDA receptor (ATD) control adsorbent (NMDAR (ATD) control adsorbent).

[0074] An anti-NMDA receptor antibody (AssayGenie, HUFI01510) was added to a solution containing each of the aforementioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4° C. The next day, the adsorbent was precipitated by centrifugation and washed with buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and then the anti-NMDA receptor antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0075] The amount of anti-NMDA receptor antibody in the collected solution was measured using an anti-NMDA receptor antibody measurement kit (AssayGenie, HUFI01510), and the antibody adsorption rate for each adsorbent was calculated, with the amount of antibody added being 100%. As a result, the anti-NMDA receptor antibody adsorption rate for the NMDAR orientation-controlled adsorbent was 2.2 times that of the NMDAR control adsorbent (Table 1).

[0076] Example 10: Antibody adsorption experiment using an anti-PLA2R antibody adsorption column Anti-PLA2R antibodies are known to be autoantibodies involved in the onset and progression of membranous nephropathy. The amino acid sequence information for human membrane phospholipase A2 receptor (PLA2R) is as follows: NCBI Accession No. NP_031392.3 UniprotKB Q13018 Full length: 1463 amino acids Signal peptide: amino acid numbers 1-20 Mature peptide: amino acid numbers 21-1463 Transmembrane domain: amino acid numbers 1398-141 Extracellular domain: amino acid numbers 21-1397

[0077] The autoantigenic fragment of PLA2R used was the CysR domain (amino acid numbers 38-161, KGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEY (SEQ ID NO: 5)) described in the reference (Front. Immunol. 15:1335998. https: / / doi.org / 10.3389 / fimmu.2024.1335998).

[0078] A PLA2R expression vector was constructed by inserting a sequence containing a GGGGS linker, an Avi tag, and a His tag into the C-terminus of the CysR domain of PLA2R, whose binding ability to anti-PLA2R antibodies has already been demonstrated, into the pcDNA3.4 TOPO vector (Invitrogen, A14697). The constructed expression vector was transfected into Chinese Hamster Ovary cells (CHO cells), and the protein expressed in the culture supernatant was purified using a nickel chelate column. The Avi tag site of the purified protein was biotinylated using biotin ligase (Avidity, BIRA) to produce biotinylated PLA2R (CysR). Biotinylated PLA2R(CysR) was immobilized on avidin agarose (Thermo Fisher Scientific, 29200) via avidin-biotin binding to prepare a PLA2R(CysR) orientation-controlled adsorbent.

[0079] The membrane-type PLA2R (CysR) protein was immobilized on Cellufine Formyl (JNC, 19853) by covalent bonding via an amino group to prepare a PLA2R (CysR) control adsorbent.

[0080] An anti-PLA2R antibody (EUROIMMUNE JAPAN, EA 1254-9601 G) was added to a solution containing each of the aforementioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4°C. The next day, the adsorbent was precipitated by centrifugation and washed with buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and then the anti-PLA2R antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0081] The amount of anti-PLA2R antibody in the recovered solution was measured using an anti-PLA2R antibody assay kit (EUROIMMUNE JAPAN, EA 1254-9601 G), and the antibody adsorption rate for each adsorbent was calculated, with the amount of antibody added being 100%. As a result, the anti-PLA2R antibody adsorption rate for the PLA2R orientation-controlled adsorbent was 1.9 times that of the PLA2R control adsorbent (Table 1).

[0082] Example 11: Antibody adsorption experiment using anti-THSD7A antibody adsorption column Anti-THSD7A antibodies are known to be autoantibodies involved in the onset and progression of membranous nephropathy. The extracellular domain of human thrombospondin type 1 domain-containing 7A (THSD7A) was used as the autoantigen fragment. The amino acid sequence information for THSD7A is as follows: NCBI Accession No. NP_056019.1 UniprotKB Q9UPZ6 Full length: 1657 amino acids Signal peptide: amino acid numbers 1-47 Transmembrane domain: amino acid numbers 1608-1628 Extracellular domain: amino acid numbers 48-1607 (autoantibody-binding region)

[0083] Thrombospondin type 1 domain-containing 7A protein (LifeSpan Biosciences Inc., LS-G95931-20) with a Myc tag attached to its C-terminus was immobilized on Anti-Myc-tag mAb-Magnetic Agarose (Medical and Biological Laboratories, M047-10) via the Myc tag to prepare a thrombospondin type 1 domain-containing 7A orientation-controlled adsorbent (THSD7A orientation-controlled adsorbent).

[0084] The thrombospondin type 1 domain-containing 7A was immobilized to Cellufine Formyl (JNC, 19853) by covalent bonding via an amino group to prepare a thrombospondin type 1 domain-containing 7A control adsorbent (THSD7A control adsorbent).

[0085] An anti-THSD7A antibody (CLOUD-CLONE CORP WUHAN, AEN909HU) was added to a solution containing each of the above-mentioned adsorbents, and the mixture was mixed by inversion for 12 hours at 4°C. The next day, the adsorbent was precipitated by centrifugation and washed with a buffer (50 mM Tris, 150 mM NaCl, pH 7.2), and then the anti-THSD7A antibody bound to the adsorbent was recovered using an acidic solution (0.1 M glycine buffer, pH 2.8).

[0086] The amount of anti-THSD7A antibody contained in the recovered solution was measured using an anti-THSD7A antibody measurement kit (CLOUD-CLONE CORP WUHAN, AEN909HU), and the antibody adsorption rate of each adsorbent was calculated, assuming the amount of antibody added as 100%. As a result, the anti-THSD7A antibody adsorption rate of the THSD7A orientation-controlled adsorbent was 1.5 times that of the THSD7A control adsorbent (Table 1).

[0087]

Claims

1. A complex comprising an autoantigen protein or its antibody-binding fragment that is specifically bound by a disease-specific autoantibody, and a substrate, in which the autoantigen protein or fragment is immobilized on the surface of the substrate with controlled orientation.

2. The complex of claim 1, wherein the autoantigenic protein is selected from the group consisting of desmoglein 3, desmoglein 1, muscle-specific receptor tyrosine kinase, nicotinic acetylcholine receptor, glomerular basement membrane type 4 collagen, BP180, BP230, aquaporin 4, myelin oligodendrocyte glycoprotein, NMDA receptor, membrane-type phospholipase A2 receptor, and thrombospondin type 1 domain-containing 7A.

3. The complex according to claim 1 or 2, wherein the autoantigen protein or fragment is immobilized on the surface of the substrate in a controlled orientation via an avidin-biotin bond, an ionic bond, a chelate bond, a covalent bond, or an antigen-antibody bond.

4. The conjugate of claim 3, wherein said avidin is deglycosylated avidin.

5. The complex according to any one of claims 1 to 4, wherein the substrate is a solid carrier or support.

6. A disease-specific autoantibody adsorbent comprising the complex according to any one of claims 1 to 5.

7. The disease-specific autoantibody adsorbent according to claim 6, for use in the treatment or prevention of a disease selected from the group consisting of pemphigus, myasthenia gravis, anti-glomerular basement membrane nephritis, pemphigoid, neuromyelitis optica, anti-NMDA receptor encephalitis, and membranous nephropathy.

8. A column comprising the disease-specific autoantibody adsorbent according to claim 6.

9. The column according to claim 8, which is for use in apheresis.

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