Apheresis column for the treatment of rheumatoid arthritis

Abstract

This provides a novel method for treating rheumatoid arthritis that is highly effective and does not cause side effects that could harm patients. [Solution] The present invention provides an apheresis column loaded with a solid support containing a composition comprising at least one peptide selected from the group consisting of a specific sequence, and the composition.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising one or more citrullinated peptides, and citrullinated peptides. Apheresis column having a solid support containing a composition, and citrullinated peptide composition Alternatively, using an apheresis column containing the composition, anti-citrullinated protein autoantibodies Regarding methods for treating autoimmune diseases. [Background technology]

[0002] Rheumatoid arthritis (RA) is the most common human autoimmune disease and the most common chronic inflammatory disease. It is rheumatoid arthritis. This disease affects 0.5 to 1% of the population in developed countries and is a chronic joint and It is characterized by destructive inflammation. Rheumatoid arthritis is caused by an antiimmune globulin called "rheumatoid factor". This involves the production of antibodies and anti-citrullinated peptide / protein antibodies (ACPA). CPA often appears several years before the onset of symptoms (Non-Patent Literature 1), and occurs 70-80% of the time before RA develops. It is observed in % (Non-Patent Literature 2). ACPA is highly specific and present in the early stages of the disease. ACPA has high diagnostic value. ACPA is often associated with more severe progression and has a relative prognosis. It has. Finally, it can also predict the evolution of undifferentiated arthritis to RA, so It has measured values. ACPA is a type of ELISA (enzyme-linked immunosorbent assay), It can be detected by an immunoassay using a microtiter plate. (Medical procedure) A test classically used is a commercially available test called anti-CCP (anti-cyclic citrullinated peptide). This is a test. The test developed and used by the inventors is citrullinated fibrino The AhFibA test (anti-human fibrinogen antibody) detects antibodies that recognize the enzyme. The results of AhFibA and anti-CCP trials are generally consistent, but 5-10% of patients experience symptoms. There is a discrepancy.

[0003] As soon as the disease is diagnosed, in order to reduce the risk of progression to irreversible joint damage, Various specific treatments are prescribed to a greater or lesser extent.

[0004] After corticosteroids, immunosuppressants have become the first-line treatment prescribed to most patients. These include resistant forms of the disease, such as anti-TNF-α, anti-IL6R, and anti-CD20. Various biotherapies are sometimes initiated in succession. All of these treatments are continued chronically. It must be done, and is not always effective, or is only effective for a limited time, Furthermore, all of them have more or less serious side effects. Finally, in some patients, this treatment Despite these measures, the disease remains resistant and progresses. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Rantapaa-Dahlqvist S, et al.Arthritis Rheum.2003 Oct;48(10):2741-9, Nielen MM,et al.Arthritis Rheum.2004 Feb;50(2):380-6 [Non-Patent Document 2] De Rycke L,et al.Ann.Rheum.Dis.(2004 63:12,1587-1593,2004) [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, it is highly effective and does not cause side effects that harm patients, R Finding new ways to treat A is still necessary. [Means for solving the problem]

[0007] In recent years, as demonstrated by the inventors, citrullinated phytophosphates present in the joints of patients The role of ACPA in the development and maintenance of arthritis through the formation of immune complexes with brinogen. This is widely recognized by the international community. These immune complexes are various effectors —Through this mechanism, it induces the secretion of pro-inflammatory cytokines, particularly TNF-α, and joint inflammation It induces and maintains the synthesis of ACPA.

[0008] The inventors have discovered that citrullinated human fibrin exhibits an immunodominant property, known as ACPA dominance. Citrullinated peptides are pre-targets and therefore can specifically bind to them. The inventors identified the peptides that can remove pathogenic ACPA from the patient's body. The inventors came up with the idea of ​​removing the given citrullinated peptide. By using this column, ACPA can be purified from patient plasma to a near 100% purity. We found that it can be obtained. Next, the inventors found that anti-citrullinated drugs such as rheumatoid arthritis We designed a new device to treat autoimmune diseases caused by protein autoantibodies.

[0009] Therefore, the object of the present invention is -α171 having amino acid sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 8) -185 Cit This peptide is called a peptide in which X1 and X2 each contain a citrulline residue. Peptides that represent -α6 having amino acid sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) 21-635 Cit A peptide called X1, X2, and X3 are each citrull Peptides representing lyl residues -β6 having amino acid sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 15) 0-74 Cit-NH2 This peptide is called citrulline peptide, and X1 and X2 are each citrulline peptides. This represents a citrullyl residue where X3 has a carboxamide group instead of a carboxyl group. Peptides representing conductors, -Has the amino acid sequence GPX1VVEX2HQSACKDS (SEQ ID NO: 6) Ac -α 36-50 Cit It is a peptide called, in which the N-terminal G residue is acetylated, Peptides and / or amino acid sequences GP in which X1 and X2 each represent citrulline residues. α36-50 having X1VVEX2HQSACKDS (Sequence ID 5) Cit It is called A group of peptides in which X1 and X2 each represent a citrullyl residue. A solid support containing a composition comprising at least one peptide selected from is loaded. It is a ferresis column, One or more of these peptides are immobilized directly or indirectly on a support, Regarding resis columns.

[0010] The present invention also, -α171 having amino acid sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 8) -185 Cit This peptide is called a peptide in which X1 and X2 each contain a citrulline residue. Peptides that represent -α6 having the amino acid sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) 21-635 Cit A peptide called, wherein X1, X2 and X3 are each citrulline A peptide representing a residue, - having an amino acid sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 15) β6 0-74 Cit-NH2 A peptide called, wherein X1 and X2 are each citrulline A residue, and X3 represents a citrullyl derivative having a carboxamide group instead of a carboxyl group A peptide representing a conductor, - having an amino acid sequence GPX1VVEX2HQSACKDS (SEQ ID NO: 6) Ac -α 36-50 Cit A peptide called, wherein the N-terminal G residue is acetylated, X1 and X2 each represent a citrulline residue peptide, and / or an amino acid sequence GPX 1VVEX2HQSACKDS (SEQ ID NO: 5) having α36-50 Cit A peptide called A peptide, wherein X1 and X2 each represent a citrulline residue peptide selected from the group consisting of A composition comprising at least two peptides selected.

[0011] The present invention also relates to an apheresis column, preferably for treating autoimmune diseases caused by anti-citrullinated Protein autoantibodies, such as rheumatoid arthritis, in the production of an apheresis column The use of the composition according to the present invention.

[0012] The present invention also relates to the treatment or prevention of autoimmune diseases caused by anti-citrullinated protein autoantibodies, particularly rheumatoid Arthritis, preferably for treatment or prevention by apheresis, for use in the A composition according to the present invention, wherein one or more peptides of the composition are immobilized on a solid support A composition. [Modes for carrying out the invention]

[0013] Detailed description of the invention composition The present invention -α171- having amino acid sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 8) 185 Cit This peptide is called a peptide in which X1 and X2 each represent a citrulline residue. S peptide, -α62 having the amino acid sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) 1-635 Cit A peptide called X1, X2, and X3 are each citrulline peptides. Peptides representing residues -β60 having the amino acid sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 15) -74 Cit-NH2 This peptide is called citrulline, where X1 and X2 are respectively citrulline This represents a residue where X3 is a citrullyl derivative having a carboxamide group instead of a carboxyl group. Peptides that represent the body, -Has the amino acid sequence GPX1VVEX2HQSACKDS (SEQ ID NO: 6) Ac -α3 6-50 Cit It is a peptide called X, in which the N-terminal G residue is acetylated. Peptides in which 1 and X2 each represent citrullyl residues, and / or amino acid sequence GPX1 α36-50 containing VVEX2HQSACKDS (Sequence ID 5) Cit Pep Select from the group consisting of peptides in which X1 and X2 each represent a citrullyl residue. The present invention relates to a composition comprising at least one selected peptide.

[0014] Various sequences are summarized in Table 1 below. [Table 1] TIFF2026090253000002.tif39170

[0015] The compositions according to the present invention also include derivatives or fragments of the peptides defined above. Peptide derivatives, for example, facilitate their synthesis and / or improve their stability. It can have modifiers intended to be beneficial.

[0016] Examples of such derivatives include amides in which the carboxyl group is converted to an esterified or amide group. It is a peptide containing anoacids. β60-74 Cit-NH2 In the case of peptides, the latter derivative This is the β60 amino acid sequence of SEQ ID NO: 14, in which the C-terminal citrullyl residue is not amidated. -74 Cit It may also be a peptide called

[0017] The peptide derivatives according to the present invention also include those in which the amino group is, for example, methylated or homocitrullinated. It may contain peptides that include carbamylated or acetylated amino acids.

[0018] One example is α36-50 Cit It is an N-terminal acetylated derivative of a peptide. Ac -α36- 50 Cit It is a peptide. Peptide α36-50 Cit or Ac -α36-50 Cit These can be used as variants of each other in the present invention. However, Ac -α36- 50 Cit Peptides are preferred.

[0019] The composition according to the present invention is α171-185 Cit , α621-635 Cit, β60- 74 Cit-NH2 and Ac -α36-50Cit and / or α36-50 Cit from At least 1, 2, 3, 4, or 5 peptides, or derivatives thereof, selected from the group It can contain fragments.

[0020] Preferably, the composition according to the present invention is Ac -α36-50 Cit , α171-185 C it , α621-635 Cit and β60-74 Cit-NH2 Selected from the group consisting of It contains at least one, two, three, or four types of peptides, or their derivatives or fragments. can.

[0021] The composition according to the present invention is α171-185 Cit , α621-635 Cit , β60- 74 Cit-NH2 and α36-50 Cit and / or Ac -α36-50 Cit from One, two, three, four, or five peptides, or their derivatives or fragments, selected from the group. It can include.

[0022] Preferably, the composition according to the present invention is Ac -α36-50 Cit , α171-185 C it , α621-635 Cit and β60-74 Cit-NH2 Selected from the group consisting of It contains 1, 2, 3, 4, or 5 types of peptides, or their derivatives or fragments.

[0023] More preferably, the composition according to the present invention comprises four peptides.Ac -α36-50 C it , α171-185 Cit , α621-635 Cit and β60-74 Cit-NH Includes 2.

[0024] More preferably, the composition according to the present invention Ac -α36-50 Cit , α171- 185 Cit , α621-635 Cit and β60-74 Cit-NH2 It consists of.

[0025] The composition according to the present invention also, Ac -α36-50 Cit , α171-185 Cit , α 621-635 Cit and β60-74 Cit-NH2 1, 2 selected from the group consisting of It may consist of three or four types of peptides, or derivatives or fragments thereof.

[0026] The composition according to the present invention has an amino acid sequence S in which X1 and X2 each represent a citrulline residue. α501-515 has GIGTLDGFX1HX2HPD (Sequence ID 10) Cit and The peptide referred to as, or its derivatives or fragments, may further be included. Depending on the form, the composition is Ac -α36-50 Cit and / or α36-50 Cit , α 171-185 Cit , α621-635 Ci β60-74 Cit-NH2 and

[0027] α501~515 Cit At least 1, 2, 3, 4, 5 are selected from the group consisting of is six kinds of peptides or a peptide of one, two, three, four, five or six kinds of peptides, or a derivative or fragment thereof, and / or consists of them.

[0028] The reactivity profile of ACPA varies among patients. Therefore, the inventors found that most of the plasma samples tested were reactive Cit-NH2 to one or more of β60-74 Ac and / or α36-50. Cit showed reactivity.

[0029] Also, according to one embodiment, the composition according to the present invention comprises at least β60-74 Cit- NH2 and / or Ac -α36-50 Cit peptide (alternatively α36-50 Cit ).

[0030] However, there is plasma that is not reactive to these peptides but is reactive to other peptides of the present invention. Therefore, in order to cover the maximum number of types of plasma reactivity profiles, it is preferable to combine as many peptides as possible. Therefore, preferably, the composition according to the present invention comprises at least α171-185

[0031] Cit α621-635, Cit β60-74 Cit-NH2 and Ac -α36-50 Cit peptide (alternatively α36-50 Cit ). More preferably, the composition comprises α1 71-185 Cit α621-635, Cit β60-74 Cit-NH2 and Ac - α36-50 Cit Peptides (or alternatively, α36-50) Cit ) and in some cases α50 1-515 Cit Contains peptides.

[0032] The present invention also relates to an apheresis column, preferably any anti-citrullinated protein auto In the manufacture of apheresis columns for treating or preventing autoimmune diseases with antibodies Regarding the use of the composition defined in the above. Autoimmunity by anti-citrullinated protein autoantibodies. The disease is selected from the group consisting of Sjögren's syndrome, juvenile idiopathic arthritis, and rheumatoid arthritis. It is possible. Autoimmune diseases caused by anti-citrullinated protein autoantibodies are preferable. It is rheumatoid arthritis.

[0033] Apheresis Column The present invention also relates to a method in which one or more peptides are immobilized directly or indirectly on a support. This relates to an apheresis column loaded with a solid support containing the composition defined above.

[0034] The term "load" refers to a column where a liquid such as blood or blood products comes into contact with a solid support. This means that the column supports or contains a solid support so that it can flow through it. do.

[0035] Generally speaking, apheresis is the reinjection of blood or blood products from a subject back into the subject. A medical procedure that modifies the latter by adding, removing, and / or substituting one or more components beforehand. This is a technology that involves circulation outside the body via a medical device. More specifically, plasmapheresis Apheresis is when the product processed by apheresis is plasma. In particular, Rituximab has been described to desensitize ABO-incompatible kidney transplant candidates, and these candidates had anti-ABO antibodies that compromised the tolerance of ABO-incompatible grafts (L. Rostaing et al., Treatment of large plasm a volumes using specific immunoadsorptio n to desensitize ABO-incompatible kidney transplant candidates, J. Nephropathology . 2016;5(3):90-97).

[0036] Thus, the apheresis column according to the present invention enables the removal of ACPA from the subject's blood or blood products such as plasma. In conventional apheresis methods, blood is directly collected at the level of the subject's vein or artery.

[0037] In some embodiments, the blood is separated into one or more blood products (e.g., solid fractions including red blood cells and white blood cells, platelets, and liquid fractions such as plasma). Components (e.g., autoantibodies) are removed from one of the blood products (e.g., plasma). Finally, the blood products are combined. The blood can then be reinjected into the subject's vein or artery.

[0038] The present invention particularly relates to - a peptide having the amino acid sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 8) of α171- 185 Cit where X1 and X2 each represent a citrullyl residue, - a peptide having the amino acid sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) of α62 1-635 CitA peptide called X1, X2, and X3 are each citrulline peptides. Peptides representing residues -β60 having the amino acid sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 15) -74 Cit-NH2 This peptide is called citrulline, where X1 and X2 are respectively citrulline This represents a residue where X3 is a citrullyl derivative having a carboxamide group instead of a carboxyl group. Peptides that represent the body, -Has the amino acid sequence GPX1VVEX2HQSACKDS (SEQ ID NO: 6) Ac -α3 6-50 Cit It is a peptide called X, in which the N-terminal G residue is acetylated. A peptide and / or amino acid sequence GPX in which 1 and X2 each represent a citrullyl residue. α36-50 having 1VVEX2HQSACKDS (Sequence ID 5) Cit The Pe From the group consisting of peptides in which X1 and X2 each represent a citrulline residue, Select at least one, at least two, at least three, at least four, or fewer At least five types, or one, two, three, four, or five types of peptides, or their derivatives or fragments. an apheresis column loaded with a solid support containing a composition comprising, One or more of these peptides are immobilized directly or indirectly on a support, Regarding resis columns.

[0039] The solid support of the apheresis column also represents citrulline residues, with X1 and X2 respectively. α501 has the amino acid sequence SGIGTLDGFX1HX2HPD (SEQ ID NO: 10) -515 Cit It may contain a peptide called, or a derivative or fragment thereof.

[0040] More preferably, the solid support of the apheresis column is four types of peptides. Ac -α 36-50 Cit , α171-185 Cit , α621-635 Cit and β60-74 Cit-NH2 Includes.

[0041] The present invention also includes at least one, at least two, at least three, at least four or This column includes at least 5 types, or 1, 2, 3, 4, or 5 types of apheresis columns. Regarding the assembly, each apheresis column is: -α171- having amino acid sequence VDIDIKIX1SCX2GSCS (SEQ ID NO: 8) 185 Cit This peptide is called a peptide in which X1 and X2 each represent a citrulline residue. S peptide, -α62 having the amino acid sequence X1GHAKSX2PVX3GIHTS (SEQ ID NO: 12) 1-635 Cit A peptide called X1, X2, and X3 are each citrulline peptides. Peptides representing residues -β60 having the amino acid sequence X1PAPPPISGGGYX2AX3 (SEQ ID NO: 15) -74 Cit-NH2 This peptide is called citrulline, where X1 and X2 are respectively citrulline This represents a residue where X3 is a citrullyl derivative having a carboxamide group instead of a carboxyl group. Peptides that represent the body, -Has the amino acid sequence GPX1VVEX2HQSACKDS (SEQ ID NO: 6) Ac -α3 6-50 Cit It is a peptide called X, in which the N-terminal G residue is acetylated. A peptide and / or amino acid sequence GPX in which 1 and X2 each represent a citrullyl residue. α36-50 having 1VVEX2HQSACKDS (Sequence ID 5) Cit The Pe From the group consisting of peptides in which X1 and X2 each represent a citrulline residue, A solid support containing a selected peptide, or its derivative or fragment, is loaded. One or more of these peptides are immobilized directly or indirectly on a support.

[0042] According to one embodiment, the apheresis column assembly is, Ac -α36-5 0 Cit Peptides, α171-185 Cit Peptide, α621-635 Cit peptide and β60-74 Cit-NH2 Four types of affine loaded with a solid support containing peptides. Includes a resis column. According to one embodiment, the apheresis column assembly is α50 1-515 Cit It can contain even more peptides.

[0043] The peptide according to the present invention is preferably a synthetic peptide.

[0044] The peptide according to the present invention can be biotinylated by methods known in the prior art. Yes, it is possible. In certain embodiments, one or more peptides are connected to biotin via spacer groups. The spacer allows the citrullinated peptide to be separated from the solid support, and A This facilitates recognition by CPA. The binding properties of the peptide according to the present invention to ACPA. Any suitable spacer that allows for maintenance can be used in this invention. In a particular embodiment, the spacer is made of polyethylene glycol (PEG) or aminohexane. It is an acid (AHX).

[0045] The solid support for immobilizing the peptide of the present invention is known in the prior art. The body support is preferably made from a material that does not activate blood cells. It is preferable to use a support that has been treated, such as a heparinized support. Alternatively, the patient The blood can be treated with an anticoagulant such as heparin before application to the support.

[0046] The solid support is a polysaccharide, preferably a high molecular weight polymer, for example, a polymer of 100 kDa or more, e.g. For example, it can be made from agarose or cellulose. The polysaccharide of the support is crosslinked. It may be crosslinked, or it may not be. Other polystyrenes such as carboxylated polystyrene A magnetic material can be used. The solid support may be in the form of magnetic beads or glass. stomach.

[0047] The solid support may be porous or non-porous. The solid support may be spherical or non-porous. The particles may be regular in shape or in a specific form. The average particle size is preferably 10 μm to 2 mm. The size may be in the range of 30 μm to 100 μm.

[0048] The support can be treated with an anticoagulant such as heparin.

[0049] Methods for immobilizing peptides on solid supports are known. Therefore, the present invention relates to The peptide can be immobilized directly or indirectly onto a support.

[0050] α171-185 Cit , α621-635 Cit , and β60-74 Ci t-NH2 Also, depending on the case, α501-515 Cit Selected from the group consisting of peptides One or more peptides, directly or indirectly, are attached to a solid support by their N-terminus. Fixed and / or - Ac -α36-50 Cit and / or α36-50 Cit Peptides have their C-terminus It is immobilized directly or indirectly onto a solid support by this means.

[0051] Preferably, α171-185 Cit , α621-635 Cit and β60-74 Ci t-NH2 The peptides inside, and in some cases α501-515 Cit Peptides are It is immobilized directly or indirectly on a solid support by its N-terminus, Ac -α36-50 C it Peptides (or alternatively, α36-50) Cit ) is on a solid support via its C-terminus It is fixed directly or indirectly.

[0052] Indirect immobilization can be performed using appropriate intermediate compounds. A preferred method for immobilization is to use biotin and avidin or streptavidin, etc. This is based on the interaction with the intermediate compound. Therefore, it is necessary for the biotinylation and solid support of the peptide. The use of avidin or streptavidin immobilized on the body allows the peptide to be transferred to a solid support. It can be reliably attached. Specifically, this method allows the peptide according to the present invention to bio Provided in a tinned form, with streptavidin (or alternatively avidin) immobilized on the surface. To provide a solid support that has been modified, and one or more biotinylated peptides according to the present invention This may include bringing an aqueous solution into contact with a support, and rinsing the support with an aqueous solvent. ru.

[0053] In a preferred embodiment, the solid support is directly immobilized thereon. Contains vidin or avidin. One or more Ac -α36-50 Cit Peptides (or substitutes) Alternatively, α36-50 Cit ), α171-185 Cit , α621-635 Cit and β 60-74 Cit-NH2 , and in some cases α501-515 Cit Peptides in Biot It is converted to C and linked to streptavidin (or avidin). The citrullinated peptide is C Biotinylation can be performed at the terminal or N-terminus. Preferably, α171-185 C it , α621-635 Cit and β60-74 Cit-NH2 peptides, as well as in some cases Furthermore, the α501-515Cit peptide is biotinylated at its N-terminus. Ac -α36 -50 Cit Peptides (or alternatively, α36-50) Cit ) has biotin at its C-terminus It will be transformed. Ac -α36-50 Cit Peptides (or alternatively, α36-50) Cit ), α 171-185 Cit , α621-635 Cit and β60-74 Cit-NH2 ,Line up Depending on the case, α501-515 Cit Peptides have spacer groups, such as aminohexanoic acid. Biotinylation can be performed via (AHX) or, alternatively, via PEG. Biotin and Avidin In addition to affinity with γ or streptavidin, antibody-antigen interactions also affect the support. It can be used for indirect immobilization of peptides onto surfaces.

[0054] Alternatively, for example, the primary sequence of a peptide onto a solid support activated with cyanide bromide Direct immobilization of peptides via mino functional groups, or solid support containing activated epoxides. Direct immobilization of peptides via carboxyl functional groups in the primary sequence of peptides, etc. Immobilizing peptides directly onto a solid support using ioconjugation technology. This can be done. Alternatively, the peptide can be immobilized on a solid support using a "click" chemical reaction. The peptide and support can have appropriate interreactive chemical functional groups (azide and aluminum). Modified with (Kin). In other embodiments, Staudinger ligation chemical reaction Using a suitable derivative solid support, appropriately modified peptides are immobilized. It is possible.

[0055] Processing method The present invention also applies to apheresis columns, preferably anti-citrullinated protein autoantibodies. Defined for use in apheresis columns for treating or preventing autoimmune diseases. The above-mentioned composition is relating to the above.

[0056] Autoimmune diseases caused by anti-citrullinated protein autoantibodies occur in the body of one person, and the same person This is a disease characterized by the expression of antibodies against citrullinated proteins. Autoimmune diseases caused by autoantibodies include Sjögren's syndrome, juvenile idiopathic arthritis, and joint problems. It can be selected from the group consisting of rheumatoid arthritis. Preferably, anti-citrullinated proteins. An example of an autoimmune disease caused by autoantibodies is rheumatoid arthritis.

[0057] The present invention also relates to autoimmune diseases caused by anti-citrullinated protein autoantibodies, preferably related to It is used in the treatment or prevention of rheumatoid arthritis, preferably in the treatment or prevention by apheresis. A composition as defined above herein, wherein one or more of the compositions This invention relates to a composition in which a butylene is immobilized on a solid support.

[0058] Preferably, the solid support is loaded onto the apheresis column.

[0059] Preferably, the treatment or prevention of autoimmune diseases by anti-citrullinated protein autoantibodies is The blood of a patient suffering from the autoimmune disease is taken in a set as defined above in this specification. The solid support containing the product is treated in vitro using an apheresis column loaded with the product. This includes the following.

[0060] The present invention also relates to an apheresis column, preferably for the treatment of rheumatoid arthritis. The above-mentioned composition is for use in cis-columns.

[0061] The present invention also relates to the use of the apheresis column defined above, particularly for anti-citrullinated proteins. This treatment or prevention of autoimmune diseases caused by chlorine autoantibodies, more preferably rheumatoid arthritis Regarding the use of apheresis columns.

[0062] The present invention also relates to apheresis-based treatment or prognosis in subjects requiring apheresis. A method for prevention, in which the apheresis column defined above is used. .

[0063] The present invention also involves the extraction of anti-citrullinated peptide antibodies (ACPA) from target blood or blood products. Regarding methods for purifying, - To provide the apheresis column defined above, -This column is used to deplete the target blood or blood product from ACPA. This includes contact with blood products.

[0064] The subject of this invention is preferably mammals, and more preferably humans.

[0065] In a preferred embodiment, the subject treated by the present invention is a severe form of rheumatoid arthritis. The subject possesses high-titer anti-citrullinated protein / peptide antibodies (A Subjects with CPA may also be included. High-titer ACPA subjects are subject to the AhFibA trial. This is defined as a subject with an OD of ≥ 0.9 in ACPA administration. (AhFibA trial) In this context, an OD of ≥ 0.9 corresponds to the 40% of rheumatoid arthritis patients with the highest rate of ACPA. (Figure 1).

[0066] The present invention will be further explained by the following figures and examples. However, these examples and figures are This should not be interpreted as limiting the scope of the present invention. [Brief explanation of the drawing]

[0067] [Figure 1] This figure shows the distribution of AhFibA titers measured in serum samples from 202 AhFibA-positive rheumatoid arthritis patients. The optical density threshold for separating quartiles (corrected OD defines the titer) is indicated. [Figure 2] This figure shows the percentage of ELISA-reactive serum for four peptides—β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit, and α171-185Cit—from serum derived from 202 AhFibA-positive rheumatoid arthritis patients. [Figure 3]This figure shows the distribution of AhFibA-positive sera from 202 rheumatoid arthritis patients based on their reactivity profiles to four peptides: β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit, and α171-185Cit. [Figure 4] This figure shows the correlation between antibody titers against four peptides—β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit, and α171-185Ci—in the serum of 202 tested individuals. At each intersection, the Spearman coefficient and the correlation (p) are recorded. [Figure 5] To simultaneously purify anti-β60-74Cit-NH2, anti-Ac-α36-50Cit, anti-α621-635Cit, and anti-α171-185Cit antibodies, two types of chromatography were used: (A) a series-connected monopeptide column, or (B) a single column containing a balanced mixture of four monopeptide matrices. [Figure 6] This figure shows chromatography performed on a pool of serum from RA patients using either four consecutively mounted columns loaded with β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit, and α171-185Cit peptides (left histogram), or a column containing a mixture of the four peptides (right histogram). Pool 97 consisted of 97 serum samples of the same volume from patients with typical elevated ACPA titers in the RA patient population, while Pool 27 consisted of 27 serum samples of the same volume from patients with high ACPA titers (OD≧1.5). The pools (start serum: SD - black bar), non-retained fraction (FNR - white bar), and eluate (gray bar) were analyzed using AhFibA ELISA (n≧4), and the results are expressed as corrected OD. [Figure 7]This figure shows the method for calculating the purification rate of serum after chromatography. The non-retaining fraction is analyzed by AhFibA ELISA, and the resulting OD is compared with that obtained from serial dilutions of the starting serum (reference curve). The purification rate can be determined by the dilution ratio. In this example, 1 / 50 vs. 1 / 400: the amount of antibody in FNR is 8 times less, i.e., 12.5% ​​of the antibody present in the starting serum. Therefore, 87.5% of ACPA was purified. [Figure 8] This figure shows the calculation of the ACPA purification rate of serum (number 41) after chromatography using four columns loaded with β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit, and α171-185Cit peptides, respectively, mounted in series. The non-retained fraction was analyzed by AhFibA ELISA and the OD was obtained by comparing it with that obtained from the dilution of the starting serum (reference curve). The purification rate can be determined by the dilution ratio from 1 / 50 to 1 / 1718. 98% of the ACPA was purified. [Figure 9] This figure shows chromatography prepared from individual serum samples from pools 97, 27, and 10 derived from RA patients, using four columns mounted in series and loaded with β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit, and α171-185Cit peptides, respectively. For each pool and individual serum sample, the starting serum (SD - black bar) and non-retaining fraction (FNR - white bar) were analyzed by AhFibA ELISA (n≧4), and their titers are expressed as corrected OD. The purification rate (%) was calculated for all samples subjected to chromatography (gray bars). [Figure 10] This figure shows the correlation between the ACPA (AhFibA ELISA) titer of the starting serum (before chromatography) and the purification rate obtained after chromatography using four columns loaded with β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit, and α171-185Cit peptides, respectively. The correlation is very significant (p=0.014), indicating that higher serum ACPA titers are associated with more efficient purification. [Examples]

[0068] Materials and methods

[0069] serum sample Serum samples were prepared using citrullinated human fibrinogen as an immunoadsorbent in ACPA. The "AhFibA" ELISA test, which enables detection and titer measurement, has shown positive results in 20 This was obtained from two rheumatoid arthritis patients.

[0070] Synthetic peptides human fibrinogen α-chain residues 36-50, 171-185 and 621-635 ( NP_068657-isoform 2) and residues 60-74 of the β chain (AAA18024) A peptide corresponding to ) was synthesized.

[0071] Peptides are converted into their citrullinated forms (all arginyl residues are replaced with citrullyl residues). (By systematic substitution) or their natural non-citrullinated forms (arginyl residues) It was synthesized using [this method].

[0072] β60-74 and β60-74 Cit Peptides, instead of terminal carboxyl groups, Synthesized using boxamide, β60-74 NH2 and β60-74 Cit-NH2 Pepu I obtained Chido.

[0073] Using acetyl, α36-50 and α36-50 Cit The peptide is synthesized at the N-terminus, Ac -α36-50 and Ac -α36-50 Cit We obtained a peptide.

[0074] Peptides, Ac -α36-50 Cit Regarding peptides, aminohexanoyl sulfate At the C-terminus of α171-185 Cit , α621-635 Cit and β60-74 Ci t-NH2 For the peptide, biotinylation was performed at the N-terminus of the same spacer.

[0075] Test ELISA PBS (1.5 mM KH2PO4 SIGMA 79) at concentrations of 5 and 10 μg / mL 5488, 7mM K2HPO4SIGMA P3786, 0.15M NaClS Antigen or immunoadsorbent (citrullinated f) solubilized in IGMA 31434 (pH 7.2) (Ibrinogen or citrullinated peptides and non-citrullinated peptides) overnight at 4°C By incubating, microtiter plate wells (MAXISORP) Adsorb onto NUNC 2023-09), or add 5 μg / mL of avidin in PBS. Incubate overnight at °C, then wash, and incubate at 4°C with 10 μg / mL in PBS. Use in the avidin-biotin system, where biotinylated peptides are added after incubation for a certain period of time. Block in PBS 2% BSA (Sigma A3059) at 4°C for 1 hour. Then, wash in PBS 0.1% Tween(registered trademark) 20 (SIGMA P1379). After purification, the sample to be tested is immersed in PBS 2% BSA 2M NaCl at a ratio of 1:50 (Av). Precipitate with a din-biotin system at a ratio of 1:100 or equivalent corrective dilution. After washing, the secondary antibody (Goat anti-human IgG Fc fragment coupled to horseradish peroxidase, So Incubate Uthern Biotech 2040-05) at 4°C for 1 hour. After washing, citric acid / H3PO4 buffer pH 5 (0.05M Sigma C075 citrate) 9. Ortho-Fen in 0.1M Na2HPO4 (Prolabo 28026292) Didiamine dihydrochloride (Sigma P2788), 0.03% H2O2 (Sigma a) Use a solution of H1009) to develop the color at room temperature for 5 minutes, then use a 6N H2SO4 solution. Stop at (Sigma 07208). Multiskan Fc plate reader ( Optical density (OD) is read at 492 nm using ThermoScientific. .

[0076] Conditions for conducting the ELISA test [Table 2]

[0077] Expression of ELISA test results: For peptides, the reactivity of citrullinated peptides and non-citrullinated peptides. The variation in OD between the reactivity of the trullinated peptide and the reactivity of each reactivity was taken into consideration, and each reactivity was pre-bra Subtract it from the Σ (OD in the absence of the sample). This yields the following equation.

[0078]

number

[0079] Furthermore, the non-citrullinated form of fibrinogen does not show reactivity, therefore it is not evaluated. There is no need to do so. Therefore, in the AhFibA trial, the OD value is simply taken from the blank. Subtract (OD in the absence of the sample).

[0080] The ΔDO of the peptide and the OD of citrullinated fibrinogen indicate the titer of the tested sample. It is thought that this represents something.

[0081] Inter-assay variability is due to the rarity of the pool of patient serum tested in each microtiter plate. This is corrected by using the range of interpretation. This reference pool, named P97, is A It consisted of 97 samples of the same volume from 97 patients with high CPA titers. The OD values ​​within a range are the average of 30 previous measurements taken from the same pool at each point within that range. It is compared to the average reference OD value. Then, using the least squares method, on the corresponding plate... A correction coefficient can be obtained that is applied to all OD values ​​obtained. This corrected OD is used in the trial. It corresponds to the potency of the material.

[0082]

number

[0083] Construction of a chromatography column Four 1mL HiTrap streptavidin HP columns (GE, 29-0513- 24) Four biotinylated β60-74 Cit-NH2 , Ac -α36-50 Cit , α621-635 Cit and α171-185 Cit Each peptide can be processed independently. Do this. After equilibrating the column with PBS, add the saturated solution of biotinylated peptide to the column volume (1 Inject (mL) at a rate of 0.2 mL / min, incubate for 15 minutes, then collect from the column. , freeze-dry.

[0084] The amount of peptide remaining in the freeze-dried fraction is determined by high-performance liquid chromatography (UPLC). This is determined by subtracting this amount from the starting amount to determine the amount of peptide bound to the column. To estimate.

[0085]

Table 3

[0086] The respective amounts of the peptides shown in the above table were loaded into four columns. After being used in the continuous purification test, β60-74 Cit-NH2 α36 Ac -50 Cit , α621-635 Cit and α171-185 Cit The matrices of the four columns loaded with the peptides were recovered, mixed, and then filled into an empty 10 mL column (GE, C10 / 10) having a volume adapter (GE, AC10) to form four mixed peptide-loaded columns with a final volume of 4 mL.

[0087] Purification Two representative pools, P97 (described above) and P27, pools of sera from 27 patients having an ACPA titer greater than 1.5, and individual sera selected based on their reactivity to 4β60-74 α36 Cit-NH2 Ac -50 Cit α621-635 Cit and α171-185 Cit were diluted 1 / 4 with PBS before chromatography.

[0088] After equilibration of the column with PBS at a flow rate of 0.5 mL / min, 6 mL volumes of the diluted pool or sera were applied to the following β60-74 peptide, then α36 Cit-NH2 Ac -50 Cit α621-635 Cit and α171-185Cit The loaded column Next, the peptides are injected into four columns mounted in series, in the order of the four peptides in each column. The fraction that was not retained was collected. After washing with PBS, it was treated with 0.2 M glycine at pH 3. Using n-HCl buffer (Invitrogen, 15527-013), four peptides were analyzed. Antibodies specific to were eluted. The eluted fraction was collected and eluted in 2M Tris solution (Eurome). The pH was adjusted to 7 using dex (77-86-1). Then, the starting serum, non-retained fraction, and eluate were used. The samples were then tested by ELISA after correction of the dilution factor induced by chromatography.

[0089] Determination of the purification ratio or rate For each treated pool or serum, residual immunoassay in the purified non-retained fraction of ACPA was performed. Responsiveness (AhFibA ELISA test) and the corresponding range of dilution of pooled or starting serum. Then, the purification ratio was calculated.

[0090]

number

[0091] A hypothetical example of the calculation of the purification rate is shown in Figure 7 (1 / 50 after chromatographic dilution correction). To obtain an OD equivalent to that of the non-retaining fraction (tested), dilute the starting serum to 1 / 400. It must be explained. Therefore, the concentration of ACPA in the non-retained fraction is equal to the concentration in the starting serum. It is 1 / 8 of the degree. 1 / 8 of the ACPA in the starting serum, or 12.5%, is in the non-retained fraction. It is not purified because it is present in the initial serum. 7 / 8 of ACPA, i.e., 87.5%. It can be estimated that a certain percentage was purified, and this is the purification rate.

[0092] Reactivity profile of serum samples Reactivity profiles of patient sera against citrullinated fibrinogen and four citrullinated peptides were determined by ELISA in at least two independent assays performed in duplicate for each of the 202 samples. For reproducibility, the inner range of the microtiter plates, which required the application of a correction factor between 0.5 and 2 relative to the reference range, was taken into account.

[0093] Results ELISA reactivity profiles of sera from rheumatoid arthritis (RA) patients against citrullinated fibrinogen (AhFibA test) and four immunodominant peptides.

[0094] Two hundred and two sera from RA patients were tested with AhFibA and ranked according to their reactivity (OD or titer) in descending order (Figure 1). According to their titers, the patients were divided into the following quartiles of low (OD = [0.056 - 0.615]), medium (OD = [0.615 - 1.161]), high (OD = [1.161 - 1.876]) and very high (OD ≥ 1.876) titers.

[0095] Figure 2 shows the percentage of RA sera reactive against each of the four peptides. 76% were positive for the β60 - 74 peptide, which was confirmed to be the most immunodominant. Cit-NH2

[0096] The individual reactivity of sera against the four peptides was highly variable, allowing the definition of numerous reactivity profiles. The frequency of the different profiles observed is shown in Figure 3. Of these 202 sera, 78% were reactive against at least two of the four peptides. ​​​​​​​​​​​​Furthermore, 95% react with at least one of the four peptides. These results indicate that chromatography The use of a combination of four peptides in Raffy has shown a very broad spectrum of patient needs. This indicates that it should be possible to target them.

[0097] By exploring the correlation between serum reactivity to various peptides, β60-74 Cit-N H2 , α621-635 Cit and α171-185 Cit Significant correlations between peptides It has become clear that these correlations reflect a certain degree of cross-reactivity between these peptides. However, their relative weakness is that each peptide possesses its own reactivity. , α36 Ac -50 Cit No correlation was observed between this peptide and the other three peptides. (Figure 4) The dual input table shows the Spearman coefficients calculated for all peptide pairs compared and their These results indicate whether or not the findings are significant. These results will be applied to ACPA in as many patients as possible. To develop a purification system that targets the entire molecule, all four peptides must be used together. This supports the idea that it is necessary. Therefore, ultimately, the choice is that this be done. That is the case.

[0098] β60-74 Cit-NH2 , Ac -α36-50 Cit , α621-635 Cit and α171-185 Cit The peptide is loaded and four cells are attached in series. Lammagraphy columns, or columns containing a mixture of four peptides, from RA patients Purification of serum pool.

[0099] Two chromatography systems, as shown in Figure 5, were devised and used with four types of peptides. ACPA was purified from the patient's serum. The first system used four types of β60-74 Ci t-NH2 , Ac -α36-50 Cit , α621-635 Cit and α171-185 Cit (A) Connecting four columns in series, each loaded with one peptide. Therefore, the second system involves mixing four monopeptide matrices in equal amounts and then combining them. This consists of loading into a single column (B).

[0100] The effectiveness of two systems was compared using two pools of serum derived from RA patients. These conditions were chosen because they are more stringent than the conditions for testing individual serums. In fact, in a pool containing many serums with different reactivity profiles, ACPA The mixture is highly polyclonal and consists of ACPAs with different antigen specificities. The first pool, P97, has an AhFibA titer distribution that is representative of the general population of RA patients. It consists of 97 serum samples. The second pool, P27, has a high AhFibA titer (OD>1). 5) Consists of 27 serum samples derived from patients.

[0101] Figure 6 shows β60-74 respectively. Cit-NH2 , Ac -α36-50 Cit , α621 -635 Cit and α171-185 Cit Four peptides are mounted in series. In either a filtered column or a column containing a mixture of four peptides, these poodles The results of chromatography performed on the column are shown. A significant proportion of ACPA was found in the column. The remaining portion is absorbed and can be eluted from the column, but the other portion that is not absorbed remains in the column. It is found in fractions that are not retained by the chromatography system. Both chromatography systems are Poo It is effective in purifying most of the ACPA contained in the ur, and little difference is obtained. It doesn't seem to exist.

[0102] Furthermore, four types of β60-74 Cit-NH2 , Ac -α36-50 Cit , α621-6 35 Cit and α171-185 Cit Based on the reactivity profile to peptides Ten ACPA-positive serum samples were selected (Table 4 below).

[0103] [Table 4]

[0104] These serums, along with two pools P97 and P27, were each taken using 4β60-74. Cit -NH2 , Ac -α36-50 Cit , α621-635 Cit and α171-185 C it The procedure was performed using four serial columns loaded with peptides. For all samples, ACPA was used. The titer decreased sharply after passing through the column (Figure 9).

[0105] To compare results between different chromatography methods and samples, an efficiency index for purification is used. It was necessary. The optical density of the sample in ELISA is the amount of ACPA contained in this sample. Since it correlates exponentially rather than linearly, the titer of the starting serum and the amount not retained in the column The simple ratio of optical density between the titers of the fractions could not be used to calculate the purification rate.

[0106] Therefore, the inventors have demonstrated a series of dilutions of the starting serum in AhFibA ELISA. Using a graph method that allows determining the OD of an object and establishing a reference curve After correcting for chromatography-related dilution factors, the OD of the non-retained fraction is similarly determined. The graph readings on the reference curve correspond to which dilution of the starting serum the OD of the non-retained fraction represents. This indicates the ratio of purification to the experimental dilution of the non-retained fraction and the corresponding dilution of the starting serum. This is the result calculated from [the data].

[0107] Figure 7 shows the graph-based method for determining the purification rate. One of the processed serums (serum) An example of how to calculate this percentage for number 41) is shown in Figure 8.

[0108] The sperm count was calculated for two pools and for 10 individual serum samples after they had passed through the column once. All yield rates were over 65%, and 7 out of 12, including 2 pools, were over 85%. In the case of set number 41, this percentage reaches 98%.

[0109] ACPA titer of the starting serum and the result after passing through four columns mounted in series. The relationship with the purification rate was investigated. Figure 10 shows a strong and significant correlation between these two parameters. This indicates the presence of Spearman's r: 0.70, p=0.014. Serum ACPA titer. The higher the titer, the more efficient the purification. This counterintuitive result suggests that high-titer ACPA has affinity This is due to the fact that the higher viscosity allows it to bind to the antigen more efficiently.

[0110] References Throughout this application, various references describe the prior art to which the present invention belongs. The disclosure of the references is incorporated by reference into this application.

Claims

1. - Amino acid sequence VDIKIX １ SCX ２ α171 having GSCS (Sequence ID 8) -185 Ｃｉｔ It is a peptide called X １ and X ２ Each of them has a citrullyl residue Peptides that represent - Amino acid sequence X １ GHAKSX ２ PVX ３ α6 having GIHTS (Sequence ID 12) 21 - 635 Ｃｉｔ a peptide called １ , X ２ and X ３ are each citrulline Peptides representing lyl residues - Amino acid sequence X １ PAPPPISGGGYX ２ AX ３ β6 having (Sequence ID 15) 0-74 Ｃｉｔ－ＮＨ２ It is a peptide called X １ and X ２ Each of them is citrull X represents a residue, ３ Citrullyl derivative having a carboxamide group instead of a carboxyl group Peptides representing conductors, - Amino acid sequence GPX １ VVEX ２ HQSACKDS (Sequence ID 6) Ａｃ -α 36-50 Ｃｉｔ It is a peptide called, in which the N-terminal G residue is acetylated, X １ and X ２ Peptides and / or amino acid sequences GP, where each represents a citrullyl residue. X １ VVEX ２ α36-50 having HQSACKDS (Sequence ID 5) Ｃｉｔ It is called It is a peptide, X １ and X ２ Each of these represents a peptide containing a citrullyl residue. A solid support comprising a composition containing at least one peptide selected from the group consisting of the following: A loaded apheresis column, One or more of these peptides are immobilized directly or indirectly on the support, Feresis column.

2. α３６－５０ Ｃｉｔ 、 Ａｃ －α３６－５０ Ｃｉｔ 、α１７１－１８５ Ｃｉｔ 、α６２１ -635 Ｃｉｔ and β60-74 Ｃｉｔ－ＮＨ２ At least two selected from the group consisting of The apheresis column according to claim 1, characterized by containing a peptide.

3. a171-185 Ｃｉｔ ,α621-635 Ｃｉｔ ,b60-74 Ｃｉｔ－ＮＨ２ ムプ Chido, and α36-50 Ｃｉｔ and / or Ａｃ -α36-50 Ｃｉｔ This contains peptides An apheresis column according to claim 1 or 2, characterized by the following:

4. Amino acid sequence SGIGTLDGFX １ HX ２ HPD (Sequence ID 10) α501-51 5 Ｃｉｔ It is a peptide, X １ and X ２ Each of these peptides represents a citrullyl residue. An apheresis column according to any one of claims 1 to 3, characterized by containing [a certain substance].

5. -α171-185 Ｃｉｔ , α621-635 Ｃｉｔ , and β60-74 Ｃｉｔ－ＮＨ ２ Also, depending on the circumstances, α501-515 Ｃｉｔ One selected from the group consisting of peptides or Multiple peptides are immobilized directly or indirectly onto the solid support by their N-terminuses. transformed, and / or - Ａｃ -α36-50 Ｃｉｔ and / or α36-50 Ｃｉｔ Peptides are their C-terminus The claim is characterized by being fixed directly or indirectly onto the solid support by its ends. An apheresis column as described in any one of items 1 to 4.

6. The one or more peptides are transferred onto the solid support by one or more intermediate compounds. The after part according to any one of claims 1 to 5, characterized in that it is indirectly fixed to Election column.

7. - Amino acid sequence VDIKIX １ SCX ２ α171 having GSCS (Sequence ID 8) -185 Ｃｉｔ It is a peptide called X １ and X ２ Each of them has a citrullyl residue Peptides that represent - Amino acid sequence X １ GHAKSX ２ PVX ３ α6 having GIHTS (Sequence ID 12) 21-635 Ｃｉｔ It is a peptide called X １ , X ２ and X ３ Each of them is Citol Peptides representing lyl residues - Amino acid sequence X １ PAPPPISGGGYX ２ AX ３ β6 having (Sequence ID 15) 0-74 Ｃｉｔ－ＮＨ２ It is a peptide called X １ and X ２ Each of them is citrull X represents a residue, ３ Citrullyl derivative having a carboxamide group instead of a carboxyl group Peptides representing conductors, - Amino acid sequence GPX １ VVEX ２ HQSACKDS (Sequence ID 6) Ａｃ -α 36-50 Ｃｉｔ It is a peptide called, in which the N-terminal G residue is acetylated, X １ and X ２ Each of these represents a peptide and / or amino acid sequence GPX, where each represents a citrullyl residue. １ VVEX ２ α36-50 having HQSACKDS (Sequence ID 5) Ｃｉｔ The Pe Petit de, X １ and X ２ Each of these represents a peptide containing a citrullyl residue. A composition comprising at least two peptides selected from the group consisting of the following.

8. A composition according to any one of claims 1 to 7 in the production of an apheresis column use.

9. The composition has one or more peptides immobilized on a solid support, anti-cyto For use in treating or preventing autoimmune diseases caused by phospholipid protein autoantibodies. The composition according to any one of claims 1 to 7.

10. The aforementioned autoimmune disease caused by anti-citrullinated protein autoantibodies is Sjögren's syndrome. Selected from the group consisting of juvenile idiopathic arthritis and rheumatoid arthritis, preferably rheumatoid arthritis. A composition for use according to claim 9, characterized in that it is a composition for use according to claim 9.

11. The treated subject has high titer anti-citrullinated protein autoantibodies (ACPA). A composition for use according to claim 9 or 10, characterized by the following:

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