WO2008016134A1

WO2008016134A1 - Method for predicting prognosis of rheumatoid arthritis patients

Info

Publication number: WO2008016134A1
Application number: PCT/JP2007/065238
Authority: WO
Inventors: Norihiro Nishimoto; Jun Hashimoto
Original assignee: Norihiro Nishimoto; Jun Hashimoto
Priority date: 2006-08-04
Filing date: 2007-08-03
Publication date: 2008-02-07
Also published as: JPWO2008016134A1; JP5330829B2

Abstract

In patients treated with MRA, PIIANP decreased at week 12 from the start of the treatment compared with before the treatment, and it was found that the progression of joint destruction was suppressed in such patients, specifically, there was a correlation between the change in PIIANP at week 12 and the change in JSN at week 52. From this, it was found that by measuring PIIANP in the treatment of RA and examining its change over time in the early phase of the treatment, the prognosis of RA patients by the treatment can be predicted.

Description

Specification

Prediction method for treatment prognosis in patients with rheumatoid arthritis

Technical field

[0001] The present invention relates to a method for predicting the prognosis of treatment of rheumatoid arthritis using the amount of PIIANP in a subject who has been treated for rheumatoid arthritis.

Background art

[0002] Cartilage has a cartilage interstitium and has voids for receiving chondrocytes.

The physiological characteristics of cartilage are those of the matrix, including collagen, elastin and acidic mucus polysaccharides. Collagen and elastin are flexible substances, and the hardness of the intercellular substances in cartilage is due to the combined proteodalycan. Chondrocytes mainly produce collagen (type II collagen).

[0003] Typell collagen is synthesized as a procollagen molecule containing N- and C-propeptides (それぞれ and PIICP, respectively) at each end. Of the procollagens produced by RNA splicing, TypellA procollagen is a molecule that contains a cysteine-rich globular domain encoded by exon 2 in sputum.

[0004] Rheumatoid arthritis, which is chronic in rheumatoid arthritis, is a systemic chronic disease in which abnormal proliferation of connective tissues such as synovial tissue is observed in the joints. In the joints of rheumatoid arthritis patients with chronic rheumatoid arthritis, the synovial cells are proliferated prominently, the formation of a multilayered structure due to abnormal synovial cell proliferation (pannus formation), Infiltration, angiogenesis into synovial cells, and infiltration of inflammatory cells such as lymphocytes and macrophages are observed. Involvement of various site force-in and growth factors has been reported as a mechanism of the onset of rheumatoid arthritis, which is chronic in rheumatoid arthritis.In patients with rheumatoid arthritis who have chronic rheumatoid arthritis, -1, IL_8, TNF-a, TGF-β, FGF, PDGF, and other site force-in or growth factors are detected. IL-1 and TNFα are considered to be potent synovial growth factors (Non-patent Documents 1 to 3), and synovial cells produce IL-6 by stimulation with IL-1 and TNF. (Non-Patent Document 4). Furthermore, it has been reported that anti-IL-6 antibody is useful as a therapeutic agent for rheumatoid arthritis (Patent Document 1). Anti-IL-6 receptor It has also been reported that antibodies are useful as therapeutic agents for rheumatoid arthritis (Patent Document 2).

[0005] To date, as a marker of Typell collagen synthesis, the procollagen type ΠΑ N-propeptide (hereinafter referred to as ΡΠΑΝΡ) in patients with OA (osteoarthritis) at baseline (before treatment) has been measured. Reported! /, Ru (Non-Patent Document 5).

Furthermore, it has been reported that the level of serum sputum is reduced in patients with knee OA and RA (Non-patent Documents 6 and 7).

[0006] However, in comparison with the healthy control group at the baseline, it was stated that the patient group had a lower vagina level! /! If you are not actually investigating the changes over time and use ΡΠΑΝΡ as an index to monitor the therapeutic effect of a drug, it is inferred that the lower the value, the better the therapeutic effect! Cannot be derived. Furthermore, it was pointed out that further research is needed to determine whether PIIANP can be used as an early stage marker for OA and RA (Non-patent Document 8).

Yes

[0007] Prior art document information related to the invention of the present application is shown below.

Patent Literature l: WO96 / 11020

Patent Document 2: JP-A-8208514

Non-Patent Document 1: Thornton et al., Clin. Exp. Immunol. 86: 79-86, 1991

Non-Patent Document 2: Lafyatis et al., J. Immunol. 143: 1142-1148, 1989

Non-Patent Document 3: Gitter et al., Immunology 66: 196-200, 1989

Non-Patent Document 4: Ito et al., Arthritis Rheum. 35: 1197-1201, 1992

Non-Patent Document 5: Patrick G. et al., ARTHRITIS & RHEUMATISM 46 (10), 2002, pp 261

3-2624

Non-Patent Document 6: Rousseau J. C. et al., OsteoArthritis and Cartilage (2004) 12, 440-44 7

Non-Patent Document 7: Rousseau J. C. et al., ARTHRITIS & RHEUMATISM 43 (9), 2000 Non-Patent Document 8: K.A. Elsaid, CO. Chichester, Clinica Chimica Acta 365 (2006) 68-7 7

Disclosure of the invention Problems to be solved by the invention

[0008] The present invention has been made in view of such a situation, and an object thereof is to predict the prognosis of treatment of rheumatoid arthritis using the amount of PIIANP as an index in a subject who has undergone rheumatoid arthritis treatment. It is to provide a method.

Means for solving the problem

[0009] In order to solve the above-mentioned problems, the present inventors have conducted intensive research.

MRA (Tocilizumab: anti-IL-6 receptor antibody, W092 / 19759) analysis of phase III randomized controlled trials for rheumatoid arthritis (hereinafter also referred to as RA) analyzed cartilage metabolic markers PIIAN P, known as a type II collagen synthesis marker), decreased in the 12th week from the start of treatment compared to before treatment. Specifically, the 12-week change in PIIANP amount is correlated with the 52-week change in Joint space narrowing (JSN), which is an indicator of joint destruction.

[0010] To date, RA patients have lower PIIANP levels compared to healthy individuals! /, And have been reported! / If the progression of destruction is suppressed, it is expected that the PIIANP level will increase, but the results of this study analysis show that the prognosis is better if a decrease in the PIIANP level is observed at the beginning of treatment. From the technical level, the opposite knowledge was obtained.

[0011] It was found that the prognosis of RA patients by this treatment can be predicted by measuring the amount of PIIANP in the treatment of RA and examining the changes over time in the early stage of the treatment. The invention has been completed.

In addition, the 12-week change in MMP-3 correlates with the 52-week change in JSN and erosion, while the two biochemical markers show changes independent of each other. By combining JSN and erosion prognosis prediction by measuring the amount of MMP-3, a more accurate prediction of joint destruction prognosis is possible.

[0012] More specifically, the present invention provides the following [1] to [; 12].

[1] A method for predicting a prognosis for treatment of rheumatoid arthritis in a subject who has undergone rheumatoid arthritis treatment, comprising the following steps (a) to (c): (a) Subject power Sample collection process

(b) Measuring the amount of procollagen type IIA N-propeptide (PIIANP) in the collected sample

(c) The process of predicting the prognosis for treatment of rheumatoid arthritis in subjects using the amount of PIIANP as an index

[2] The method according to [1], wherein, in the step (c) according to [1], when the amount of PIIANP decreases compared to before the start of treatment, the prognosis by the treatment is predicted to be good.

[3] The method according to [2], wherein when the amount of PIIANP is reduced at an early stage after the initiation of rheumatoid arthritis treatment, the prognosis by the treatment is predicted to be good.

[4] The method according to [2] or [3], wherein whether or not the prognosis of treatment for rheumatoid arthritis is good is determined by whether or not the progress of joint destruction in rheumatoid arthritis is suppressed. [5] The method according to [1], wherein a sample is collected from a subject over time and the amount of PIIANP is measured.

[6] The method according to [1] to [5], wherein the rheumatoid arthritis is early rheumatoid arthritis.

[7] The method according to [1], wherein the sample collected from the subject is plasma, serum, urine, or joint fluid.

[8] The method according to [1], wherein the rheumatoid arthritis treatment is treatment by administration of an anti-rheumatic agent.

Yes

[9] Rheumatoid arthritis treatment is IL-1 inhibitor, IL-6 inhibitor, TNF-a inhibitor, IL-15 inhibitor

Or the method according to [1], which is treatment by administration of an IL-17 inhibitor.

[10] The method according to [1], wherein the rheumatoid arthritis treatment is treatment by administration of an antibody that inhibits IL-1, IL-6, TNF-a, IL-15 or IL-17 signal transmission.

[1 1] Rheumatoid arthritis treatment is anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-TNF-a antibody, anti-C

Treatment with administration of D20 antibody, methotrexate or CTLA4-IgG fusion protein

The method according to [1].

[12] A kit for predicting the prognosis of treatment for rheumatoid arthritis, comprising a reagent for measuring the amount of PIIANP. [0013] [Embodiment of the Invention]

The present inventors have found that it is possible to predict the prognosis of RA patients due to the treatment by measuring the amount of soot in the treatment of RA and examining the change over time in the early stage of the treatment. The present invention is based on these findings.

The present invention relates to a method for predicting a prognosis for treatment of rheumatoid arthritis in a subject who has undergone rheumatoid arthritis treatment.

[0015] In the present invention, the treatment of rheumatoid arthritis includes treatment by administration of an anti-rheumatic agent, anti-cytokine therapy, a drug for CD20 effective for malignant lymphoma, an antibiotic, an immunosuppressant, hyperlipidemia Administration of therapeutic agents for cerebral dysfunction, therapeutic agents for multiple myeloma, etc., or treatment by hematopoietic stem cell transplantation. In the present invention, the anti-rheumatic drug includes all drugs used for the treatment of rheumatoid arthritis. Preferably, for example, disease-modifying anti-rheumatic drugs (DM ARDs), IL-1 inhibitors (IL-1 signaling) Such as low molecular weight compounds having such action, including anakinra), STAT-3 inhibitors, IL-6 vaccines, IL-6 fusion proteins, IL-6 inhibitors (IL- 6 drugs that inhibit signal transduction, such as low molecular weight compounds having such actions, including anti-IL-6 receptor antibodies (such as tocilizumab) and anti-IL-6 antibodies), TNF-a inhibitors (including Drugs that inhibit TNF-a signaling include, for example, low molecular weight compounds having such effects, chimeric anti-TNF-α monoclonal antibody, infliximab, TNFR2-IgGFc fusion protein, etanercebut, human anti-TNF-α Monochroma Antibodies include adalimumab, other certolizumab pegol, Golim umab, etc.), IL-15 inhibitors (including drugs that inhibit IL-15 signaling, such as low molecular weight compounds such as AMG714) IL-17 inhibitor (including drugs that inhibit IL-1 7 signal transduction, including low molecular weight compounds having such action, receptor antibodies, ligand antibodies, etc.), anti-CD20 antibodies (Such as rituximab), 2-penicillamine derivatives, Roxithromycin T cell costimulatory modulators (such as CTLA4_Ig fusion protein abatacebut), AP-1 inhibitors, lipopolysaccharide binding proteins (LBPs), complement inhibitors (such as Eculizumab) ), Protein (p38) kinase inhibitors, lipid A analogs (such as E-5531), melanin analogs and the like. As the rheumatoid arthritis therapeutic agent of the present invention, particularly preferred is anti-site force-in therapy, specifically Treatment includes administration of an IL-1 inhibitor, an IL-6 inhibitor, a TNF-α inhibitor, an IL-15 inhibitor, or an IL-17 inhibitor. Treatment with methotrexate, anti-CD20 antibody and CTLA4-Ig fusion protein is also preferred! /.

In the present invention, rheumatoid arthritis includes early rheumatoid arthritis. Currently, the American College of Rheumatology (ACR) classification criteria are commonly used worldwide as a diagnostic method for rheumatoid arthritis. However, if it is less than 6 weeks after the onset of rheumatoid arthritis, it is impossible to make a definitive diagnosis of the disease based on this criterion.In order to diagnose rheumatoid arthritis early, diagnosis of early rheumatoid arthritis within 1 year of onset The purpose of this study is to use the early diagnostic criteria of the Japan College of Rheumatology.

[0017] In the present invention, "treatment prognosis for rheumatoid arthritis" preferably indicates a joint destruction prognosis, but the joint destruction prognosis includes a functional prognosis, a life prognosis, a quality of life (QOL) life, Since it is deeply involved in pain prognosis, according to the present invention, it is useful for prediction of all of these listed treatment prognoses.

In the present invention, “predicting the prognosis of treatment” refers to predicting whether or not the prognosis of rheumatoid arthritis is improved by treatment. Specifically, whether the prognosis of rheumatoid arthritis has a good prognosis, life prognosis, quality of life, pain prognosis, joint destruction prognosis, etc., preferably joint prognosis is good. Further, in the present invention, “predicting the prognosis of treatment” can be rephrased as “inspecting whether or not the prognosis of rheumatism is improved by treatment”. When the subject is a human, the diagnosis of the disease is usually performed by a doctor (including those who have received instructions from the doctor; the same shall apply hereinafter), and the data regarding the prognosis of rheumatoid arthritis obtained by the present invention is Force that is useful for diagnosis by the method The method of the present invention may be such that a person other than a doctor collects and presents data useful for diagnosis by a doctor.

Furthermore, in the present invention, “predicting the treatment prognosis” can be rephrased as “indicating the treatment prognosis for a predetermined period in the patient treatment plan from the start of rheumatoid arthritis treatment”. For example, in the Examples of the present application, the measurement result of the biochemical marker at 12 weeks after the treatment shows that it is possible to predict the treatment prognosis about one year after the start of the treatment (52 weeks). [0020] The progress of joint destruction can be evaluated by the method described in Examples. Specifically, X-ray parity (Sharp score), transition of cartilage metabolic markers, correlation between X-ray changes and changes in bone and cartilage-related metabolic markers, multiple The progress of joint destruction is evaluated by analysis.

In the present invention, as a criterion for determining whether or not the joint destruction prognosis is good, the evaluation based on the sharp score described in the examples is used as one standard. The sharp score is calculated by the sum of the erosion score (Example, Table 2) and the joint-space narrowing score (JSN score), ERA column, ¾3). (Examples, Table 1). If the sharp score decreases, the progression of joint destruction in rheumatoid arthritis is suppressed, and it is judged that the prognosis for treatment of rheumatoid arthritis is good.

[0022] The method of the present invention includes a step of collecting a subject force sample. Samples collected from the subject include, but are not limited to, plasma, serum, urine, or joint fluid. Preferred are plasma and serum, and more preferred is serum.

The step of collecting a sample from the subject of the present invention is also referred to as a step of providing a sample collected from the subject.

[0023] The method of the present invention includes a step of measuring the amount of a cartilage metabolism marker in a collected sample. Examples of cartilage metabolism markers include matrix metaprotease 3 (ΜΜΡ-3) and Ρ ΙΙΑΝΡ. ΜΜΡ-3 is produced from cells such as synovial cells and chondrocytes, and is involved in remodeling of extracellular collagen fibers and destruction of cartilage collagen by activating other extracellular matrix protease family (MMPs) I think that. MMP-3 is thought to represent an inflammatory condition of the synovium. Acupuncture is a propeptide corresponding to the N-terminal part of type IIA procollagen produced during the synthesis of type II collagen (Fig. 1).

[0024] The most preferred marker for cartilage metabolism of the present invention is PIIANP. The amount of PIIANP can be measured by a known method. For example, the amount of PIIANP can be measured by ELISA, for example, U.S. Pat.No. 5,780,240, the aforementioned non-patent document 2, and Methods in Molecular medicine, Vol. 101 (Cartilage and Osteoarthritis, vol. 2: Structure and In vivo Analysis ), pp.25-38, 2004, edited by F. De Ceuninck et al. It can be measured by a method according to this.

[0025] The method of the present invention includes a step of predicting a treatment prognosis of rheumatoid arthritis in a subject using a cartilage metabolism marker as an index. In the present invention, when the amount of cartilage substitution marker changes compared to before the start of treatment, it can be predicted that the prognosis by the treatment is good. When the cartilage metabolism marker is PIIANP, it can be predicted that the prognosis by the treatment is good when the amount of NP is decreased compared with that before the start of treatment. Specifically, for example, the rate of change of PIIANP concentration in serum (% change when baseline is 100) decreases by 1 to 90%, preferably 5 to 80%, more preferably 10 to 50% In this case, the prognosis can be predicted to be good.

[0026] In the present invention, it is preferable that the change in the amount of cartilage metabolism marker compared to before the start of treatment occurs early after the start of rheumatoid arthritis treatment. Early after initiation refers to within 52 weeks after the start of treatment, preferably within 48, 36, 24, 12 weeks, more preferably within 12, 6, 1 week.

[0027] The amount of cartilage metabolism marker of the present invention is preferably measured by collecting a sample from a subject over time. Measurement over time refers to measurement according to a chronological order, for example, subjects on the first or second day of treatment, or on weeks 1, 6, 12, 24, 36, 48, 52, etc. A sample is taken from and the amount of cartilage metabolism marker is measured.

[0028] In addition, according to the present invention, a more accurate prediction of joint fracture prognosis can be achieved by combining JSN prognosis prediction by measuring PIIANP amount and prognosis prediction of JSN and erosion by measuring MMP-3 amount. Is possible. In addition, measurements of other markers such as CRP may be combined with these measurements.

[0029] The rheumatoid arthritis treatment of the present invention preferably includes treatment by administration of an IL-1 inhibitor, an IL-6 inhibitor, a TNF-a inhibitor, or an IL-15 inhibitor.

[0030] The IL-1 inhibitor, IL-6 inhibitor, TNF-a inhibitor, IL-15 inhibitor, or IL-17 inhibitor used for the treatment of rheumatoid arthritis of the present invention is IL-1, respectively. Antibodies that inhibit IL-6, TNF-a, IL-15 or IL-17 signaling are preferred. Examples of antibodies that inhibit IL-1, IL-6, TNF-a, IL-15, or IL-17 signaling include anti-HI-1 antibody, anti-HI-1 receptor antibody, anti-HI-1 antibody, HI HL-6 antibody, anti-HI HL-6 receptor antibody, anti-human TNF-α antibody, anti Examples include human TNF-α receptor antibody, anti-human HL-15 antibody, anti-human HL-15 receptor antibody, anti-human HL-17 antibody or anti-human HL-17 receptor antibody.

[0031] In the present invention, an antibody that inhibits IL-1, IL-6, TNF-a, IL_15, or IL-17 signaling can be obtained as a polyclonal or monoclonal antibody using known means. As the antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced in hyperpridoma and those produced in a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. This antibody specifically binds to HI-1, IL-6, TNF-a, IL-15, IL-17 or their receptors, and suppresses the function of these site forces.

[0032] Antibodies that inhibit IL-1, IL-6, TNF-a, IL-15, or IL-17 signaling are not particularly limited as long as they bind to the protein, mouse antibodies, rat antibodies, Usagi antibody, Hedge antibody, human antibody and the like can be used as appropriate. In addition, recombinant antibodies artificially modified for the purpose of reducing the heterologous antigenicity to humans, such as chimeric antibodies, humanized antibodies, and the like can also be used. These modified antibodies can be produced using known methods.

[0033] The chimeric antibody is a mammal other than a human, for example, an antibody comprising a heavy chain and a light chain variable region of a mouse antibody and a heavy chain and a light chain constant region of a human antibody. The DNA can be obtained by ligating DNA encoding the constant region of a human antibody, incorporating it into an expression vector, introducing it into a host, and producing it.

[0034] A humanized antibody is also called a reshaped human antibody, and a complementarity determining region (CDR) of a mammal other than a human, eg, a mouse rod, is complemented with a human antibody. It is transplanted to the sex-determining region, and its general gene recombination technique is also known. Specifically, several DNA sequences designed to link mouse antibody CDRs and human antibody framework regions (FR) with overlapping portions at the ends are prepared. The oligonucleotide is synthesized from the oligonucleotide by PCR. The obtained DNA is obtained by ligating with DNA encoding a human antibody constant region, and then incorporating it into an expression vector, which is introduced into a host and produced. Linked via CDR The human antibody FR is selected such that the complementarity determining region forms a favorable antigen-binding site. If necessary, amino acid in the framework region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site.

[0035] Methods for obtaining human antibodies are also known! For example, human lymphocytes are sensitized with a desired antigen or cells expressing the desired antigen in vitro, and the sensitized lymphocytes are fused with human myeloma cells such as U266, and the desired human antibody having activity to bind to the antigen. (See Japanese Patent Publication No. 1-59878). Moreover, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen. Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable region of a human antibody can be expressed as a single-chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector having the sequence can be prepared and a human antibody can be obtained.

[0036] The antibody used in the present invention may be a conjugated antibody bound to various molecules such as polyethylene glycol (PEG), radioactive substance, and toxin! /. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field. The “antibody” in the present invention includes these conjugated antibodies.

[0037] Examples of antibodies in the present invention include low molecular weight antibodies. The low molecular weight antibody is not particularly limited as long as it includes an antibody fragment lacking a part of a full length antibody (whole antibody such as whole IgG) and has an ability to bind to an antigen. The antibody fragment of the present invention is not particularly limited as long as it is a part of a full-length antibody, but it is particularly preferable that VH includes a heavy chain variable region (VH) or a light chain variable region (VL). It is a fragment containing both VL. Specific examples of the antibody fragment include Fab, Fab ′, F (ab ′) 2, Fv, scFv (single chain Fv), and the like. To obtain such an antibody fragment, the antibody is treated with an enzyme such as papain or pepsin to produce an antibody fragment, or A gene encoding these antibody fragments may be constructed, introduced into an expression vector, and then expressed in an appropriate host cell.

[0038] The most preferred treatment for rheumatoid arthritis according to the present invention includes treatment by administration of an IL-6 inhibitor.

Examples of the IL-6 inhibitor of the present invention include anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-gpl30 antibody, IL-6 variant, soluble IL-6 receptor variant or IL-6 or Examples include IL-6 receptor partial peptides and low molecular weight substances exhibiting similar activities, but are not particularly limited. The IL-6 inhibitor of the present invention is preferably an antibody that recognizes IL-6 receptor.

[0039] The origin of the antibody in the present invention is not particularly limited, but is preferably derived from a mammal, more preferably a human-derived antibody.

[0040] The anti-IL-6 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-IL-6 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hyperpridoma and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. This antibody binds to IL-6, thereby inhibiting the binding of IL-6 to the IL-6 receptor and blocking the intracellular transmission of IL-6 biological activity.

Examples of such antibodies include MH166 (Matsuda, T. et al., Eur. J. Immunol. (1988) 18, 95 1-956) and SK2 antibody (Sato,. Et al., 21st Japanese Society for Immunology). General meeting, academic record (1991) 21, 166).

[0041] An anti-IL-6 antibody-producing hybridoma can be basically produced using a known technique as follows. That is, using IL-6 as a sensitizing antigen and immunizing it according to the usual immunization method, the obtained immune cells are fused with known parental cells by the usual cell fusion method, and by the usual screening method, It can be produced by screening monoclonal antibody-producing cells.

[0042] Specifically, the anti-IL-6 antibody can be prepared as follows. For example, HI-6 used as a sensitizing antigen for antibody acquisition is Eur. J. Biochem (1987) 168, 543-550, J. Im munol. (1988) 140, 1534-1541, or by using the IL-6 gene / amino acid sequence disclosed in Agr. Biol. Chem. (1990) 54, 2685-2688.

[0043] After the IL-6 gene sequence is inserted into a known expression vector system to transform an appropriate host cell, the target IL-6 protein is known from the host cell or culture supernatant. The purified IL-6 protein can be used as a sensitizing antigen. Also, a fusion protein of IL-6 protein and other proteins can be used as a sensitizing antigen.

[0044] The anti-IL-6 receptor antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-IL-6 receptor antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Monoclonal antibodies derived from mammals include those produced by hyperpridoma and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. This antibody binds to the IL-6 receptor, thereby blocking the binding of IL-6 to the IL-6 receptor and blocking the transmission of IL-6 biological activity into the cell.

[0045] Examples of such antibodies include MR16-1 antibody (Tamura, T. et al. Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-l antibody (Hirata, Y et al., J. Immunol. (1989) 143, 2 900-2906), AUK12-20 antibody, AUK64-7 antibody or AUK146-15 antibody (International Patent Application Publication No. WO 92-19759) . Of these, PM-1 antibody is exemplified as a preferred monoclonal antibody against HL-6 receptor, and MR16-1 antibody is exemplified as a preferred monoclonal antibody against mouse IL-6 receptor. It is done.

[0046] An anti-IL-6 receptor monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, IL-6 receptor is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cells are fused with a known parent cell by a normal cell fusion method. Thus, a monoclonal antibody-producing cell can be screened.

[0047] Specifically, the anti-IL-6 receptor antibody can be prepared as follows. For example, the human HL-6 receptor used as a sensitizing antigen for obtaining an antibody is disclosed in European Patent Application Publication No. EP 3 25474, and the mouse IL-6 receptor is disclosed in Japanese Patent Application Publication No. JP-A 3-155795. Obtained by using the prepared IL-6 receptor gene / amino acid sequence. [0048] IL-6 receptor protein is expressed on the cell membrane and separated from the cell membrane (soluble IL-6 receptor) (Yasukawa,. Et al., J. Biochem. (1990) 108, 673-676). Soluble IL-6 receptor antibody binds to the cell membrane! /, And is composed of substantially the extracellular region of IL-6 receptor, and lacks the transmembrane region or the transmembrane region and the intracellular region. Is different from membrane-bound IL-6 receptor. As the IL-6 receptor protein, any IL-6 receptor may be used as long as it can be used as a sensitizing antigen for producing the anti-IL-6 receptor antibody used in the present invention.

[0049] After the gene sequence of IL-6 receptor is inserted into a known expression vector system to transform an appropriate host cell, the target IL-6 receptor is obtained from the host cell or the culture supernatant. The protein may be purified by a known method, and this purified IL-6 receptor protein may be used as a sensitizing antigen. In addition, cells expressing IL-6 receptor or fusion proteins of IL-6 receptor protein and other proteins may be used as sensitizing antigens.

[0050] The anti-gpl30 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. The anti-gpl30 antibody used in the present invention is particularly preferably a mammal-derived monoclonal antibody. Mammal-derived monoclonal antibodies include those produced by hyperpridoma and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. This antibody binds to gpl30, thereby inhibiting the binding of IL-6 / IL-6 receptor complex to gpl30 and blocking the transmission of IL-6 biological activity into the cell.

Examples of such antibodies include the 8 \ 64 antibody (Japanese Patent Laid-Open No. 3_219894), the 4811 antibody and the 2 ^ ¾ antibody (US 5571513), the B-S12 antibody and the B-P8 antibody (Japanese Patent Laid-Open No. 8-291199). .

[0051] An anti-gpl30 monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, gpl30 is used as a sensitizing antigen and immunized according to the usual immunization method. The obtained immune cells are fused with known parental cells by the usual cell fusion method, and then monoclonal by the usual screening method. It can be produced by screening antibody-producing cells.

[0052] Specifically, a monoclonal antibody can be prepared as follows. For example, gpl30 used as a sensitizing antigen for antibody acquisition is disclosed in European Patent Application Publication No. EP 411946. Can be obtained by using the gpl30 gene / amino acid sequence disclosed in (1).

[0053] After the gene sequence of gpl30 is inserted into a known expression vector system to transform an appropriate host cell, the target gpl30 protein is obtained from the host cell or culture supernatant by a known method. The purified gpl30 receptor protein may be purified and used as a sensitizing antigen. Alternatively, gp130-expressing cells or a fusion protein of gpl30 protein and other proteins may be used as a sensitizing antigen.

[0054] The mammal to be immunized with the sensitizing antigen is not particularly limited, but it is generally preferable to select in consideration of the compatibility with the parent cell used for cell fusion. Rodent animals such as mice, rats, hamsters and the like are used.

[0055] Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, as a general method, a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitized antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, etc., and suspended, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if necessary. Preferably, it is administered to mammals several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.

[0056] After immunizing in this manner and confirming that the desired antibody level rises in the serum, immune cells are removed from the mammal and subjected to cell fusion. Preferred immune cells that are subjected to cell fusion include spleen cells.

[0057] Mammalian myeloma cells as other parental cells to be fused with the immune cells have already been known in various cell lines such as P3X63Ag8.653 (Kearney, JF et al. J. Imm nol. 1979) 123, 1548-1550), P3X63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-l (Kohler. G. and Milstein, C. Eur. J. Immunol. 19 76) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, SF et al ·, J. Immunol. Methods (1980) 35, 1-21), S 194 (Trowbridge, ISJ Exp. Med. (197 8) 148, 313- 323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) are used as appropriate.

[0058] The cell fusion between the immune cell and the myeloma cell is basically performed by a known method, for example, The method can be carried out according to the method of Lucyutain et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

[0059] More specifically, the cell fusion is performed, for example, in a normal culture medium in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used as the fusion promoter, and an auxiliary agent such as dimethyl sulfoxide can be added and used to enhance the fusion efficiency as desired.

[0060] The usage ratio of immune cells and myeloma cells is preferably, for example, 1 to 10 times as many immune cells as the myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture can be used. In addition, serum supplements such as fetal calf serum (FCS) can be used in combination.

[0061] For cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution and pre-warmed to about 37 ° C, for example, an average molecular weight of about 1000 to 6000. PEG solution is usually added at a concentration of 30-60% (w / v) and mixed to form the desired fused cell (nobridoma). Subsequently, cell fusion agents and the like unfavorable for the growth of hypridoma can be removed by repeating the operation of adding an appropriate culture solution successively, centrifuging and removing the supernatant.

[0062] The Hypridoma is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT medium is continued for a period of time, usually several days to several weeks, sufficient for the cells (non-fusion cells) other than the desired hyperpridoma to die. The usual limiting dilution method is then used to screen and clone hybridomas producing the desired antibody.

[0063] In addition to immunizing animals other than humans to obtain the above hyperidoma, human lymphocytes are sensitized in vitro with a desired antigen protein or antigen-expressing cells, and sensitized B lymphocytes are human myeloma. It is also possible to obtain a desired human antibody having a binding activity to a desired antigen or an antigen-expressing cell by fusing with a cell such as U266 (see Japanese Patent Publication No. 1-59878). In addition, antigens or antigen-expressing cells are administered to transgenic animals with a repertoire of human antibody genes. Then, a desired human antibody may be obtained according to the method described above (International Patent Application Publication No. wo

93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

[0064] The hybridoma producing the monoclonal antibody produced in this manner can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

[0065] In order to obtain a monoclonal antibody from the hyperidoma, the hyperidoma is cultured according to a normal method and obtained as a culture supernatant thereof, or the hyperidoma is administered to a mammal compatible therewith. The method of growing and obtaining ascites is adopted. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.

[0066] For example, the production of an anti-IL-6 receptor antibody-producing hybridoma can be performed by the method disclosed in JP-A-3-139293. PM-1 antibody-producing hybridoma is injected into the abdominal cavity of BALB mice to obtain ascites, and PM-1 antibody is purified from this ascites, or this hybridoma is treated with an appropriate medium such as 10% urine fetal serum. 5% BM-Condimed Hl (Boehringer Mannheim) -containing RPMI1640 medium, Hypridoma SFM medium (GIBCO-BRL), PF 、 -ΗΜ medium (GIBCO-BRL), etc. -1 antibody purification method.

[0067] In the present invention, as a monoclonal antibody, an antibody gene is cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host, and produced using a gene recombination technique. (See, for example, Borrebaeck CA. and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0068] Specifically, mRNA encoding the variable (V) region of the antibody is isolated from cells producing the antibody of interest, such as cells or hybridomas. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) etc. to prepare total RNA, and mRNA is prepared using mRNA Purification Kit (Pharmacia). In addition, it is possible to prepare mRNA directly by using QuickPrep mRNA Purification Kit (Pharmacia).

[0069] cDNA of the antibody V region is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis (by using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit, etc.) In addition, 5'_Ampli FINDER RACE it (Clontech) and PCR are used for cDNA synthesis and amplification. 5'-RACE method (Frohman, MA et al., Proc. Natl. A cad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) l, 2919-2932) The DNA fragment of interest can be purified from the obtained PCR product and ligated with vector DNA, and a recombinant vector can be prepared from this and introduced into E. coli, etc. The colony is selected to prepare a desired recombinant vector, and the base sequence of the target DNA is confirmed by a known method such as the deoxy method.

[0070] If DNA encoding the V region of the target antibody is obtained, it is ligated with DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector. Alternatively, DNA encoding the V region of the antibody may be incorporated into an expression vector containing DNA of the antibody C region.

[0071] To produce the antibody used in the present invention, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter, as described below. Next, the host cell is transformed with this expression vector and the antibody is expressed with the force S.

[0072] In the present invention, a genetically engineered antibody, such as a chimeric antibody, a humanized antibody, a human ( human) antibodies can be used. These modified antibodies can be manufactured using known methods with the power S.

[0073] The chimeric antibody is produced by ligating the DNA encoding the antibody V region obtained as described above with the DNA encoding the human antibody C region, incorporating it into an expression vector, introducing it into a host, and producing it. (See European Patent Application Publication No. EP 125023, International Patent Application Publication No. W 0 92-19759). Using this known method, a chimeric antibody useful in the present invention can be obtained. [0074] A humanized antibody is also called a reshaped human antibody or a humanized antibody, and a complementarity determining region (CDR) of a mammal other than a human, eg, a mouse antibody, is converted into a complementarity determining region of a human antibody. It is transplanted and its general genetic recombination technique is also known! /, (See European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92-19759).

[0075] Specifically, a DNA sequence designed to link a CDR of a mouse antibody and a framework region (FR) of a human antibody has an overlapping portion at the end. It is synthesized from several prepared oligonucleotides by PCR. The obtained DNA is obtained by ligating with the DNA encoding the human antibody C region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication Number EP 239400, International Patent Application Publication Number). See WO 92-19759).

[0076] As the FR of a human antibody to be ligated via CDR, one in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, the amino acid in the framework region of the variable region of the antibody may be substituted so that the complementarity-determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato,. Et al., Cancer Res. (1993) 53, 851-856).

[0077] The human antibody C region is used for the chimeric antibody and the humanized antibody. Examples of the human antibody C region include Cγ, and for example, Cγ1, Cγ2, Cγ3, or Cγ4 can be used. In addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production.

[0078] The chimeric antibody is composed of a variable region of a non-human mammal-derived antibody and a C region derived from a human antibody, and the humanized antibody is a complementarity determining region of a non-human mammal-derived antibody and a framework region derived from a human antibody. It is useful as an antibody for use in the present invention because it consists of the C region and C region and has reduced antigenicity in the human body.

A preferred specific example of the humanized antibody used in the present invention is a humanized PM-1 antibody (see International Patent Application Publication No. WO 92-19759).

[0079] In addition to the above-described methods for obtaining human antibodies, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, a variable region of a human antibody can be expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. The power to do S. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector can be prepared from the sequence and a human antibody can be obtained. These methods are already well known and can be referred to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388. .

[0080] The antibody gene constructed as described above can be expressed and obtained by a known method. In the case of mammalian cells, it can be expressed by a commonly used useful promoter, an antibody gene to be expressed, a DNA having a poly A signal operably linked to the 3 ′ downstream thereof, or a vector containing the same. For example, as a promoter / enhancer, it is possible to make a human cytomeculowinores ij phase promoter / enhancer (numan cytomegalovirus imme diate early promoter / enhancer).

[0081] Other promoters / enhancers that can be used for expression of the antibodies used in the present invention include retroviruses, polioviruses, adenoviruses, and simian viruses 40 (SV40). A promoter / enhancer derived from a mammalian cell such as human longon factor 1α (HEF1α) may be used.

[0082] For example, when the SV40 promoter / enhancer is used, the method of Mulligan et al. (Mulligan, RC et al., Nature (1979) 277, 108-114) and the HEF1 a-mouth motor / enhancer are used. In this case, it can be easily carried out according to the method of Mizushima et al. (Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18, 5322).

[0083] In the case of E. coli, a commonly used useful promoter, a signal sequence for antibody secretion

The antibody gene to be expressed can be expressed by functionally binding it. For example, examples of the promoter include _lacZ promoter and _araB promoter. _When using a _lacZ plug motor, the method of Ward et al. (Ward, ES et al., Nature (1989) 341, 5 44-546; Ward, ES et al. FASEB J. (1992) 6, 2422-2427) When the araB promoter is used, the method of Better et al. (Better, M. et al. Science (1988) 240, 1041-1043) may be followed.

[0084] As a signal sequence for antibody secretion, a pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379-4383) may be used when it is produced in the periplasm of E. coli. After isolating the antibody produced in the periplasm, the structure of the antibody is Used in refold (see, for example, WO96 / 30394).

[0085] As replication origins, those derived from SV40, poliovirus, adenovirus, ushipapilloma quinores (BPV), etc. can be used, and further expressed for amplification of gene copy number in the host cell system. The vector may include an aminoglycoside phosphotransferase (APH) gene, a thymidine kinase (TK) gene, an E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, and the like as selectable markers.

[0086] For production of the antibody used in the present invention, any production system can be used.

Production systems for antibody production include in vitro and in vivo production systems. In vitro production systems include production systems that use eukaryotic cells and production systems that use prokaryotic cells.

Yes

[0087] When eukaryotic cells are used, there are production systems using animal cells, plant cells, or fungal cells. Animal cells include (1) mammalian cells such as CHO, COS, myeloma, BH (baby hamster kidney), HeLa, Vero, etc., (2) amphibian cells such as Xenopus oocytes, or ( 3) Insect cells such as si9, si21, and Tn5 are known. As plant cells, cells derived from Nicotiana tabacum are known, and these may be cultured in callus. As fungal cells, yeasts such as Saccharomyces spp., For example, Saccharomyces cerevisiae, filamentous spiders such as Aspergillus, such as Aspergill us niger, are known. It has been.

[0088] When prokaryotic cells are used, there are production systems using bacterial cells. Known bacterial cells include E. coli and Bacillus subtilis.

An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro. Culture is performed according to a known method. For example, DMEM, MEM, RPMI1640, IMDM can be used as the culture medium, and serum supplements such as fetal calf serum (FCS) can be used in combination. Alternatively, antibodies may be produced in vivo by transferring cells into which the antibody gene has been introduced to the abdominal cavity of animals.

[0089] On the other hand, in vivo production systems include production systems using animals and production systems using plants. Is mentioned. When animals are used, there are production systems using mammals and insects. As mammals, goats, pigs, hidges, mice, mice, etc. can be used (Vic ki Glaser, SPECTRUM Biotechnology Applications, 1993). As insects, it is possible to use silkworms. When using a plant, for example, tobacco can be used.

[0090] An antibody gene is introduced into these animals or plants to produce and recover the antibodies in the body of the animals or plants. For example, an antibody gene is inserted in the middle of a gene encoding a protein inherently produced in milk such as goat / 3 casein to prepare a fusion gene. A DNA fragment containing the fusion gene into which the antibody gene is inserted is injected into a goat embryo, and the embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgene goat born from the goat that received the embryo or its progeny. To increase the amount of milk containing the desired antibody produced from the transgenic dog, hormones may be used as appropriate in the transgenic dog (Ebert, .M. Et al., Bio / Technology ( 1994) 12, 699-702).

[0091] When a silkworm is used, a silkworm is infected with a baculovirus inserted with the target antibody gene, and a desired antibody is obtained from the body fluid of this silkworm (Maeda, S. et al., Nature (1985) 315 592-594). Furthermore, when tobacco is used, the target antibody gene is inserted into a plant expression vector, such as pMON530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. This bacterium is infected with tobacco, for example Nicotiana tabacum, and the desired antibody is obtained from the leaves of this tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138) .

[0092] As described above, when an antibody is produced in an in vitro or in vivo production system, DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) are separately incorporated into an expression vector. May be transformed simultaneously, or the host may be transformed by incorporating DNA encoding the H and L chains into a single expression vector (see International Patent Application Publication No. WO 94-11523). ).

[0093] The antibody used in the present invention may be an antibody fragment or a modified product thereof as long as it can be suitably used in the present invention. For example, antibody fragments include Fab, F (ab ′) 2, Fv, or single chain Fv (scFv) in which H chain and L chain Fv are linked by an appropriate linker.

[0094] Specifically, an antibody fragment is produced by treating an antibody with an enzyme such as papain or pepsin. Or construct genes encoding these antibody fragments, introduce them into expression vectors, and express them in appropriate host cells (for example, Co, MS et al., J. Immunol. (19 94) 152, 2968—2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 17 8, 476-496, Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Lamoyi, Ε, Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al ·, Methods in Enzymology (1989) 121, 663-66, Bird, RE et al., TIBTECH (1991) 9, 132- 137).

[0095] scFv is obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988). 85, 5879-5883). The H chain V region and the L chain V region in scFv may be derived from! /, which is described as the above antibody. As the peptide linker that links the V regions, for example, any single chain peptide consisting of amino acid residues 12-19 is used.

[0096] The scFv-encoding DNA is composed of a DNA encoding the H chain or H chain V region of the antibody, and a DNA encoding the L chain or L chain V region, and the sequence of these sequences. A portion of the DNA encoding the desired amino acid sequence is amplified by PCR using a primer pair that defines both ends of the DNA, and then a DNA encoding a portion of the peptide linker and both ends thereof are respectively H chain, Obtained by combining and amplifying primer pairs that are defined so as to be linked to the L chain.

[0097] Once the DNA encoding scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method. Can be used to obtain scFv according to conventional methods.

[0098] These antibody fragments can be produced by the host by obtaining and expressing the gene in the same manner as described above. The term “antibody” as used in the present invention encompasses these antibody fragments.

[0099] As a modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used. The “antibody” referred to in the present invention includes these modified antibodies. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field! / [0100] The antibody produced and expressed as described above can be separated from the host inside and outside the cell and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed by affinity chromatography. Examples of the column used for affinity chromatography include a protein A column and a protein G column. Examples of the carrier used for the protein A column include HyperD, POROS, S-mark harose F.F. In addition, the separation and purification methods used for ordinary proteins are not limited in any way.

[0101] For example, by appropriately selecting and combining chromatography, filters, ultrafiltration, salting out, dialysis and the like other than the above-mentioned affinity chromatography, the antibody used in the present invention can be separated and purified. . Examples of chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, and the like. These chromatographies (also known as HPLC (High Performance Liquid Chromatography)) can be applied and protected. Alternatively, reverse phase HPLC may be used.

[0102] The concentration of the antibody obtained above can be measured by measuring absorbance, ELISA, or the like. In other words, when measuring absorbance, after appropriately diluting with PBS (-), measure absorbance at 280 nm and calculate lmg / ml as 1.350D. In the case of ELISA, it is possible to measure as follows. That is, add goat anti-HgG (TAG) 100 1 diluted to l ^ g / ml with 0.1 M bicarbonate buffer (ρΗ9 · 6) to a 96-well plate (Nunc) at 4 ° C.晚 Incubate and immobilize antibody. After blocking, an appropriately diluted antibody or a sample containing the antibody used in the present invention, or Hi HGG (manufactured by CAPPEL) 100 II 1 as a sample is added, and incubated at room temperature for 1 hour.

[0103] Alkaline phosphatase-labeled anti-HgG diluted 5000 times after washing (BIO SOURCE)

Add 100 1 and incubate at room temperature for 1 hour. After washing, add the substrate solution, incubate, measure the absorbance at 405 nm using MICROPLATE READER Model 3550 (manufactured by Bio-Rad), and calculate the concentration of the desired antibody.

[0104] The IL-6 variant used in the present invention is a substance that has binding activity to the IL-6 receptor and does not transmit IL-6 biological activity. That is, IL-6 variant binds to IL-6 receptor competitively with UL-6, but does not transmit IL-6 biological activity. Block transmission.

[0105] IL-6 variants are produced by introducing mutations by substituting amino acid residues in the amino acid sequence of IL-6. The origin of IL-6, which is a variant of IL-6, is not limited, but human IL-6 is preferable in consideration of antigenicity.

[0106] Specifically, the amino acid sequence of IL-6 can be determined using a known molecular modeling program such as W HATIF (Vriend et al., J. Mol. Graphics (1990) 8, 52-56). This is done by predicting the next structure and evaluating the effect on the total number of amino acid residues to be substituted. After determining the appropriate replacement amino acid residue, by introducing a mutation that replaces the amino acid by the usual PCR method, using a vector containing the base sequence encoding the HL-6 gene as a saddle type, A gene encoding the IL-6 variant is obtained. This can be incorporated into an appropriate expression vector as necessary, and an IL-6 variant can be obtained according to the expression, production and purification methods of the recombinant antibody.

[0107] Specific examples of IL-6 variants include Brakenhoif et al., J. Biol. Chem. (1994) 269, 86-93.

And Savino et al., EMBO J. (1994) 13, 1357-1367, WO 96-18648, W096-17869.

[0108] The IL-6 partial peptide or IL-6 receptor partial peptide used in the present invention has a binding activity to IL-6 receptor or IL-6, respectively, and the biological activity of IL-6 It is a substance that does not transmit activity! That is, IL-6 partial peptide or IL-6 receptor partial peptide binds to IL-6 receptor or IL-6 and captures these to specifically bind IL-6 to IL-6 receptor. Obstruct it. As a result, IL-6 does not transmit the biological activity of IL-6, thus blocking IL-6 signaling.

[0109] The IL-6 partial peptide or IL-6 receptor partial peptide is a part of the region involved in the binding between IL-6 and IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor. Alternatively, it is a peptide consisting of all amino acids. Such peptides usually consist of 10 to 80, preferably 20 to 50, more preferably 20 to 40 amino acid residues.

[0110] IL-6 partial peptide or IL-6 receptor partial peptide specifies the region involved in the binding of IL-6 to IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor A part or all of the amino acid sequence is generally known, for example, genetic engineering techniques or peptide synthesis. It can be produced by a synthesis method.

[0111] In order to prepare an IL-6 partial peptide or IL-6 receptor partial peptide by a genetic engineering technique, a DNA sequence encoding a desired peptide is incorporated into an expression vector, and expression of the recombinant antibody is performed. It can be obtained according to production and purification methods.

[0112] In order to prepare an IL-6 partial peptide or IL-6 receptor partial peptide by a peptide synthesis method, a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method may be used. it can.

[0113] Specifically, it may be carried out in accordance with the method described in the follow-up drug development Vol. 14, peptide synthesis (supervised by Haraaki Yajima, Yodogawa Shoten, 1991). As the solid-phase synthesis method, for example, an amino acid corresponding to the C-terminus of the peptide to be synthesized is bound to a support that is insoluble in an organic solvent, and the α-amino group and the side chain functional group are protected with an appropriate protecting group. By repeating the reaction of condensing amino acids one by one in the order from the C-terminal to the 末端 -terminal and the reaction of eliminating the protecting group of the α-amino group of the amino acid or peptide bound on the resin, A method of extending the tide chain is used. Solid phase peptide synthesis methods are broadly divided into Boc and Fmoc methods, depending on the type of protecting group used.

[0114] After the target peptide is synthesized in this manner, a deprotection reaction and a cleavage reaction from the peptide chain support are performed. For the cleavage reaction with the peptide chain, hydrogen fluoride or trifluoromethanesulfonic acid can usually be used for the Boc method, and TFA can be used for the Fmoc method. In the Boc method, for example, the protected peptide resin is treated in the presence of anisole in hydrogen fluoride. Next, the peptide is recovered by removing the protecting group and cleaving from the support. This is freeze-dried to obtain a crude peptide. On the other hand, in the Fmoc method, for example, the deprotection reaction and the cleavage reaction from the peptide chain support can be performed in TFA by the same operation as described above.

[0115] The obtained crude peptide can be separated and purified by applying it to HPLC. For the elution, a water-acetonitrile solvent usually used for protein purification may be used under optimum conditions. The fraction corresponding to the peak of the obtained chromatographic profile is collected and lyophilized. The peptide fraction thus purified is analyzed for molecular weight analysis, amino acid composition analysis, or amino acid sequence analysis by mass spectrum analysis. Identify by analysis.

Specific examples of the IL-6 partial peptide and the IL-6 receptor partial peptide are disclosed in JP-A-2-188600, JP-A-7-324097, JP-A-8-311098 and US Pat. No. US5210075.

[0117] The antibody used in the present invention may be a conjugated antibody bound to various molecules such as polyethylene glycol (PEG), radioactive substances, and toxins! /. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field. The “antibody” in the present invention includes these conjugated antibodies.

[0118] The present invention relates to a detection reagent for predicting a prognosis of treatment in rheumatoid arthritis, including a reagent for measuring a cartilage metabolism marker. Such detection reagents may include those used in the above-described cartilage metabolism marker measurement step. For example, an antibody, a staining solution, and the like that are required for measuring the amount of drought that is a cartilage substitution marker can be mentioned.

Furthermore, by combining the detection reagent and other elements used for detection of cartilage metabolism markers, a kit for predicting the prognosis of treatment in rheumatoid arthritis treatment can be obtained. That is, the present invention relates to a kit for predicting the prognosis of treatment in rheumatoid arthritis treatment, which contains a reagent for measuring a cartilage metabolism marker. Such a kit may contain the detection reagent described above, distilled water, salt, buffer solution, protein stabilizer, preservative and the like. In addition, for example, as a reagent for ELISA, it is possible to combine a chromogenic substrate for detecting an enzyme label and a washing solution for washing the solid phase. In addition, instructions for explaining the measurement operation can be attached to the kit.

It should be noted that all prior art documents cited in this specification are incorporated herein by reference.

Brief Description of Drawings

FIG. 1 is a schematic diagram showing the fragmentation of PIIANP and PIICP in cartilage formation.

FIG. 2 is a diagram showing a joint region of a hand whose Erosion score was measured.

FIG. 3 is a diagram showing a joint region of a hand whose JSN score was measured. FIG. 4 is a view showing a joint region of a foot where an Erosi _0n score and a JSN score were measured.

FIG. 5 is a diagram showing the results of X-ray evaluation (total shear score) of joint destruction at baseline, weeks 28 and 52.

FIG. 6 is a graph showing the results of X-ray evaluation (Erosion score) of joint destruction at baseline, weeks 28 and 52.

FIG. 7 is a graph showing the results of X-ray evaluation (JSN score) of joint destruction at baseline, weeks 28 and 52.

FIG. 8 is a graph showing changes in the amount of soot in the T group and the C group. (Α) Measurement of serum ΡΙΙΑΝΡ concentration (ng / ml), (B) Percent change from baseline in serum PIIANP concentration.

FIG. 9 shows changes in the amount of MMP-3 in T group and C group. (Α) Measurement of serum ΜΜΡ3 concentration (ng / ml) (B) Percent change (%) from baseline in serum MMP3 concentration.

[Fig. 10] A graph showing the presence or absence of an increase in Erosion in T group and the change in MMP-3 change.

[Fig. 11] A graph showing the presence or absence of an increase in Erosion in T group and the change in MMP-3 change rate.

FIG. 12 is a graph showing the presence or absence of an increase in Erosion and the change in MMP-3 change in group C.

FIG. 13 is a graph showing the presence or absence of an increase in JSN and the change in MMP-3 change rate in group T.

[FIG. 14] A graph showing the presence or absence of an increase in JSN and the transition of the wrinkle change rate.

FIG. 15 is a diagram showing the JSN ODD ratio (95% CI) after 52 weeks by multivariable logistic regression analysis.

[Fig. 16] A graph showing the Erosion ODD ratio (95% CI) after 52 weeks by multivariable logistic regression analysis.

Example

[0120] Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[0121] [Example 1] Correlation between prognosis of early rheumatoid arthritis treatment and changes in biochemical markers in MRA single administration group and subject group

Method> To determine the degree of progression of joint destruction over a year in patients with early rheumatoid arthritis treated with MRA, a humanized IL-6 receptor antibody, an X-ray evaluation was performed. In addition, initial changes in biochemical markers related to cartilage tissue, bone metabolism and inflammation were examined.

[0122] Target patients (306) were selected according to the following criteria.

(1) Disease duration 6 months or more and less than 5 years

(2) Age 20 and over

(3) There are at least lj or more DMARDs! /, Patients who are active with the administration of immunosuppressants

(4) Patients who meet the following conditions at the time of registration (within 2 weeks before study drug administration)

Pain ≥ 6 joints

Inflammatory swelling ≥ 6 joints

ESR≥30mm / hr and CRP≥2.0mg / dL

[0123] Patients were treated with group T (MRA single administration group; 8 mg / kg every 4 weeks), group C (target group; DMARDs (disease-modifying antirheumatic drugs) in general usage, and disease activation Divided into biologics and biphosphonate preparations).

Joint destruction was measured by X-ray assessment (sharp score, van der Heijde modified Sharp method) at baseline (before treatment) and at 28 and 52 weeks. The evaluation criteria are shown in the following tables;! To 3, and the joint sites (regions) where the evaluation was performed are shown in FIGS.

[0124] [Table 1]

Evaluation by Total Sharp score (total of Erosion + total of JSN) Total score (maximum) = 160+ 120+ 120 + 48 = 448 points

[Table 2] Score 0 Normal

Score 1 If they are discrete

Score 2 2 or 3 if they are larger, depending on

the surface area of the joint involved

Score 3 If the erosion is large and extends over

the imaginary middle of the bone

Score 4

Score 5 A complete collapse of the bone erosion score evaluation criteria

* If the total number of points in each joint exceeds 5 points, all points are 5 points.

(However, for ankle joints, each joint has a maximum total of 10 points)

[Table 3]

Joint space narrowing (JSN) score evaluation criteria

JSN: It generally represents cartilage!

In addition, biochemical markers (PIIANP, matrix metalloproteinase 3 (MMP-3), PIICP, osteocalcin, bone alkaline phosphatase, CRP, etc.) are measured at baseline, 24, 36, 48 and 52 weeks of treatment. did. [0128] DAS28 score: Pain (tenderness or movement pain) in joints to be observed (TJC), swollen joint pain (SJC) and ESR (erythrocyte sedimentation rate), using general evaluation by the patient (GH: general health status) (Arthritis & Rheumatism 38:44, 1995, Arthritis & Rheumatism 39:34, 1995).

28 Based on the number of joints DAS = DAS28

DAS28 = 0.56 TJC + 0.28 SJC + 0.7In ESR + 0.014 x GH

[0129] Results>

At baseline, patients had an average age of 53 years, an average disease duration of 2.3 years, an average DAS28 score of 6.5, a total sharp score of 29.4, and a CRP of 4.8 mg / dL.

By measuring changes in the total sharp score (Figure 5), bone erosion score (Figure 6), and joint space narrowing (JSN) score (Figure 7) There was significantly less progress in destruction.

In addition, 12 weeks of treatment including PIIANP (Tables 4, 6 and 8, and Figure 8), MMP-3 (Tables 5, 7 and 9, and Figure 9), PIICP, osteocalcin, bone alkaline phosphatase, and CRP. Changes in chemical markers were significantly different between T and C groups.

For PIIANP and MMP-3, measured values (ng / ml) are shown in Table 4 and Table 5, changes from baseline (ng / ml) are shown in Tables 6 and 7, and percentage change from baseline (%) Are shown in Table 8 and Table 9.

[0130] [Table 4]

eget0304_fas Summary of Bone Mineral Density and Bone and Cartiage Markers [FAS]

Visi Actual Values

TT 95% CI P- value

Mean SD SEM Med i an Min and ower-Upper (*)

S-PI IANP (NG / ML)

WEEK 0

Contro I 145 459. 210.9 17.5 409.4 124 1431 425.2 One 494.4 0.597 MRA 8mg / kg 156 447. 184.6 14.8 412.2 152 1293 418.6-477.0

WEEK 12

Control 141 467. 189. 16.0 422.2 141 1254 435.5-498.7 + 0.001 MRA 8mg / k 152 376. 151. 12.3 346.0 115 1056 352.4-400.9

WEEK 24

Control 140 470. 205.1 17.3 425.4 130 1220 436. 504. <0.001 MRA 8mg / kg 141 359. 132.6 11.2 343.7 150 1030 337. 381.

WEEK 36

Contro I 133 473. 187.6 16.3 439 161 1087 441, 505. <0.001 MRA 8mg / kg 138 358. 135.8 11.6 335. 145 1088 335. 381.

WEEK 48

Contro I 130 471. 191.3 16.8 426.0 134 1017 438. 504. <0.001 MRA 8mg / kg 135 360. 158.1 13.6 338.3 149 1420 333. 387.

WEEK 52

Control 129 471. 209,2 18.4 414.4 170 1384 434. 507. <0.001 MRA 8mg / k 134 354. 131.0 11.3 323.9 154 904 331. 376.

LastOBS

Contro I 144 472. 215.6 18.0 413.9 142 1384 437. 508. 0.00 0.001 MRA 8mg / k 157 359. 154.1 12.3 327.9 154 1420 334. 383.

Measured value of sputum (ng / ml)

n represents the number of patients contributing to summary statistics.

(*) Represents Student's t-test.

TT: Trial treatment

LastOBS: last observed value

(%) Represents the change by baseline value, which includes only patients with baseline (except 0) and summarized duration values.

[Table 5]

eget0304 fas Summary of Bone Mineral Density and Bone and Cart i I age Markers [FAS]

Visit ― Actual Va I u

TT 95% CI P- value

Mean SD SEM Med i an Lower-Upper (*)

MMP-3 (NG / ML)

WEEK 0

Control 145 408.5 233.8 19.4 334.0 59 800 370.2 ― 446.9 0.568 MRA 8mg / kg 156 393.2 232.1 18.6 330.0 61 800 356.4-429.9

WEEK 12

Control 142 332. 213.3 17. 299.0 44 800 297. 368.0 + 0.001 MRA 8mg / kg 152 165. 145.3 11. 122.0 18 800 142. 189.

WEEK 24

Control 140 306. 202.6 17. 236. 31 800 272. 339. <0.001 MRA 8mg / kg 144 121. 89.6 100. 14 505 106. 135.

20 800 261. 328. <0.001

16 368 94. 119.

WEEK 48

Control 131 294. 196.2 17.1 257. 22 800 260. 328. 0.001 MRA 8mg / kg 136 104. 86.5 78. 18 499 89. 119.

WEEK 52

Control 131 296. 195.2 17. 266. 22 800 262. 330. <0.001 MRA 8mg / kg 135 104. 88.5 74. 12 548 89. 119.

LastOBS

Control 144 298. 198.3 16. 22 800 266. 331. <0.001 MRA 8mg / k 157 106. 85.0 6.

12 548 92. 119.

Measured value of MMP-3 (ng / ml)

n represents the number of patients contributing to summary statistics.

(*) Represents Student's t-test.

TT: Trial treatment

LastOBS: last observed value

(%) Represents the change by baseline value, including only patients with baseline (except 0) and summarized duration values.

[Table 6]

Visit Change From Base to me

TT 95% CI P-value

Mean SD SEM Median Min Max Lower I Upper (*)

S-PI IANP (NG / L)

WEEK 12

Contro I 141 8.0 118.3 10.0 15.9 -545 335 -11. 27.7 0.001 M A 8mg / kg 151 -73.8 135.1 11.0 —61.2 -756 465 -95. Ί -52.1

WEEK 24

Control 140 8.7 136.1 11.5 13.4 -594 457 -14. 31.4 <0.001 MRA 8mg / kg 140 1 93.0 141.2 11.9 —93.8 -814 289 -116.-69.4

WEEK 36

Control 133 17.1 146.8 12.7 13.8 -459 575 -8. 42.3 + 0.001 MRA 8mg / kg 137 -87.1 154.4 13.2 -81.6 -828 346 -113. -61.0

WEEK 48

Control 130 16.4 146.1 12.8 3.5 -479 396 1 9. 41.7 + 0.001 MRA 8mg / kg 134 -93.7 145.4 12.6 -91.4 -847 277 -68.9

WEEK 52

Contro I 129 16. 136.1 12.0 3.5 --426 580 39.8 <0.001 MRA 8mg / kg 133 -91. 141.6 12.3 -76.8 -847 191 -115 -67.3

Contro I 144 13.5 133.2 11.1 1.2 -426 580 35.5 <0.001 MRA 8mg / k 156 -89.0 138.9 11.1 -74.9 -847 191

-67.0

Amount of change from baseline in cocoon (ng / ml)

n represents the number of patients contributing to summary statistics.

(*) Represents Student's t-test.

TT: Trial treatment

LastOBS: last observed value

[Table 7]

eget0304_fas Summary of Bone Mineral Density and Bone and Cart i I age Markers [FAS]

Vis Change From Basel ine

TT 95% CI P-value

Mean SO SEM Median Min Lower I Upper (*)

MMP-3 (NG / ML)

WEEK 12

Contro I 142 -74. 176.9 14.8 -24.5 -654 265 --104.-45.3 <0.001 MRA 8mg / kg 151 -231. 224.1 18.2 -179.2 -764 364 -267. -195.4

194.0 16.4 -46.5 -625 465 -127.-62.2

233.8 19.6 -239.1 -775 92 -316.-239.1

WEEK 36

Contro I 133 -105. 206.5 17.9 -56.0 -638 426 -141. -70.5 + 0.001 MRA 8mg / kg 139 -290. 238.0 20.2 -269.0 -780 150 --329.

WEEK 48

Control 131 -110. 213.9 18.7 -50.0 -687 451 -147. -73.3 <0.001 MRA 8mg / kg 135 -291. 243.9 21.0 -268. -781 203 -333. -250.2

WEEK 52

Contro I 131 -108. 210.6 18.4 -44.0 -684 314 -144. -71.6 <0.001 MRA 8mg / kg 134 -293. 243.6 21.0 -275.2 -780 296 -335.

LastOBS

Control 144 -107. 205.8 17.1 -44.9 -684 314 -141. -73.2 <0.001 MRA 8mg / kg 156 -286. 242.0 19.4-242, 3 -780 296 -324. -248.3

MMP-3 change from baseline (ng / ml)

n represents the number of patients contributing to summary statistics.

(*) Represents Student's t-test.

TT: Trial treatment

LastOBS: last observed value

[Table 8]

eget0304_fas Summary of Bone ineral Density and Bone and Cartilage Markers [FAS]

Visit Percent Change From Baseline

TT 95% CI P-value

Mean SD SEM Med i an Min Lower I Upper (*)

S

-9.71 <0.001 one -8.09

-10.22 to 0.001--10.75

14.90 <0.001 -9.46

13.65 <0.001 -11.84

12.84 to 0.001

-11.45

LastOBS

Control 144 6.52 31.31 2.61 0.-63 03 220. 45 36 11.68 <0.001 MRA 8mg / kg 156 -15.62 26.00 2.08 One 20 One 76 59 91. 75 -19.73 -11.51

Rate of change from baseline in PIIANP (%)

n represents the number of patients contributing to summary statistics.

(Represents Student t-test.

TT: Trial treatment

LastOBS: last observed value

(%) Represents the change due to baseline values, including only patients with baseline (except 0) and summarized duration values.

[Table 9]

eget0304_fas Summary of Bone ineral Density and Bone and Cart i I age Markers [FAS]

Percent Change From Basel ine

95% CI P-value Mean SD SEM Med i an in Max Lower I Upper (*)

MMP-3 (%)

WEE 12

Control 142 -10.18 4.04 -12.7 -18.16--2.20 +0.001 MRA 8mg / k 151 -48.51 3.00 -55.3 -54.43--42.59

WEEK 24

Control 140 -11.40 68.10 5.76 -20.4 -92.6 614.7 -22.78--0.02 <0.001 MRA 8mg / kg 143 -57.13 38.38 3.21 -69.6 -97.3 99.2 -63.47--50.78

WEEK 36

Control 133 -12.29 54.32 4.71 -16.9 -95.7 217.8 -21.61--2.97 0.001 MRA 8mg / kg 139 -59.43 41.67 3.53 -72.5 -97.6 92.4 -66.42--52.44

WEEK 48

Control 131-14.92 49.46 4.32 -16.9 -95.3 243.8 —23.47--6, 37 0.001 MRA 8mg / kg 135 -60.14 42.92 3.69 -76.4 -97.7 130.6 -67.44--52.83

WEEK 52

Control 131 -14.60 47.21 4.13 -12.8 -95.3 169.7 —22.77 1 —6.44 0.001 MRA 8mg / k 134 -60.32 45.53 3.93 -78.9 -97.5 154.4 -68.10--52.54

LastOBS

Control 144 -14.59 46.35 3.86 -14.4 -95.3 169.7 -22.22--6.95 <0.001 MRA Bmg / kg 156-59.23 45.47 3.64 -75, 5 -97.5 154.4 -66.42--52.04

o σ>

Rate of change from baseline of MMP-3 (%)

n represents the number of patients contributing to summary statistics.

(*) Represents Student's t-test.

TT: Trial treatment

LastOBS: last observed value

(%) Represents the change from baseline (excluding 0) and baseline, including only patients with summarized duration values.

[0136] The correlation between the results of X-ray evaluation of joint destruction and changes in the amount of biochemical markers is shown below.

In group T, there was a significant correlation between the rate of change in MMP-3 concentration at week 12 and the amount of change in the erosion score for one year (Spearman correlation coefficient: r = 0.23 ρ 0.01). In addition, -3-3 concentration and the change rate of ΡΠΑΝΡ concentration at the 12th week were significantly correlated with the change in JSN score for one year (ΜΜΡ-3; Γ = 0 · 28 ρ 0.01 PIIANP; r = 0.25 p 0.01).

[0137] Further, multivariate analysis was performed. As a result, the 52-week change in the Erosion score is the rate of change in the MMP-3 concentration (ρ = 0 · 03), and the 52-week change in the JSN is the 12-week rate of change in the ΜΜΡ-3 concentration (ρ 0.01) and the 12-week rate of change in sputum concentration (p 0.01) were considered appropriate (Table 10 and Table 11). In the table, “CE52” and “CJ52” represent the change from baseline in the 52nd week of Erosion score and JSN score, respectively. In the table, “PMMP312” and “ΡΡΠANP12” represent the% change when the baseline is 100 for the 12th week of MMP-3 and ΡΠΑΝΡ, respectively.

[Table 10]

Tba ^ 8 weave dure

Itodei 1IQDEL1

ftspaikdfiitt Variable: i

Analyst of Vaitiatm

of e

Sarce Df Sq 隱 ¼JBfe F ^ l & im

63. 贿 0 sues 4.

Errar m. S3 i Ί. Mm

Corre ted Total 細. 7

Hoot USE 2-76087 K 隱 β 0-0S55

Oepandant Mean 0.fldl7 »Mi R-Se 0.0451

£ {t «f Var far« E »tw Est iaates

Standard

I

Multivariate analysis (Erosion Score52)

[Table 11]

ardent: Vari ^ le-

of V ia oa

S i of Nfi

Source Square F V & i Pf> F

3 Wind 24733

Error 130 im.mm

tere ^ ted Total 133 tm. n itoot USE 3.2S3B0 0.1636

De findfi t Mean L5559T

239.0fi77S

«E

f araaete * Est mates

Varia le Df

tercet 1 .10? ^ 0.730SS

? 312 1 .m 0.00814

5 0.

: 1 0.301 ¾ m321 S

(Multivariate analysis OSN52)

[0140] The rate of change in MMP-3 concentration at 12 weeks (PMMP312) and the rate of change in PIIANP concentration (ΡΡΠΑΝΡ

12) were independent of each other and confirmed to be independent of the 12th week DAS28 score (DAS2812) (Table 12).

[0141] [Table 12]

Correlation of PPIIANP 12, DAS2812 and PMMP312

Spearman correlation coefficient

PMMP312 and DAS2812: 0.40

PPIIANP 12 and DAS2812: 0.09

PPIIANP 12 and PMMP312: 0.14

[0142] [Example 2] Correlation between progression of joint destruction and changes in biochemical markers in MRA alone administration group

Purpose>

In a study (SAMURAI STUDY) in which MRA (Tocilizumab) monotherapy group (T group) was compared with DMARDs group (C group) and examined the effect of delayed bone joint destruction for one year, group C (me an 6.1; 95% CI 4.2, 8.0) (p <0.01) (Mean 2.3; 95% CI 1.5, 3.2), indicating that signs and symptoms of improvement were exhibited (reference ARD).

Therefore, in SAMURAI STUDY, we investigated the relationship between the amount of biomarker change early after the start of treatment and the progression of joint destruction one year later, and further investigated the predictive factors.

[0143] Ku Method>

In the T group, variable selection was performed in consideration of the correlation and multicollinearity between the 12-week change in biomarker and the change in erosion score and JSN score after 1 year, and multivariate regression analysis and multiple A random mouth regression analysis was performed.

[0144] Results>

When factors affecting the change in X-ray score were examined, the 12-week change in MMP-3 was associated with a change in erosion score (R-square 0.086) (Table 13).

[Table 13] NORMALTER P-value

Explanatory variable

Evaluation

CLMMP312 1.63017 0.0075 Multiple regression analysis (bone erosion)

CLMMP312 = Fluctuation value of logarithmic variable of MMP-3 at 12th week

Changes in MMP-3, PIIANP, and CRP at 12 weeks were related to changes in JSN score (R-quare 0.186) (Table 14).

[Table 14] parameter

Explanatory variable P value

Evaluation

CLMMP312 1.65808 0.0253

CLPIIANP12 5.65204 0.0162

CLCRP12 1.13029 0.0025 Multiple regression analysis (joint space narrowing)

CLMMP312 = Fluctuation value of logarithmic variable of MMP-3 at 12th week

CLPIIANP12 = Variation value of logarithm variable of cocoon at 12th week

CLCRP12 = Change of logarithmic variable of CRP at 12th week

[0146] When comparing the 12-week change in MMP-3 by the change in Erosion score, the group with a small change in Erosion score (Erosion score 52-week change ^ 0.5), that is, a bone destruction delay effect was seen. MMP-3 (a major protease that degrades extracellular matrix components: cartilage destruction) was significantly reduced compared to the group that had no effect! (Fig. 10). The same was true for the rate of change in MMP-3 (Fig. 11). On the other hand, the same results were not seen in Group C, which is the comparison target (Figure 12).

When examined according to the amount of change in the JSN score, a significant decrease in MMP-3 was observed in the group with a small amount of change in the JSN score (JSN score: 52 weeks change: 0.5) (Figure 13). Similarly, the amount of change was small in the pupa, the group was large, and the group showed a significant decrease compared to the group (Fig. 14).

The ODD ratio was determined by multivariate logistic regression analysis. Changes in JSN after 52 weeks are predicted by changes in MMP-3 (ODD ratio: 0.333), changes in sputum (0.377), negative CRP (0.288), and changes in MMP-3 (0.485) in Erosion (Figures 15 and 16).

[0147] Based on the above results, changes in the amount of sputum and MMP-3 in the serum in the early stage of treatment should be useful as a tool for measuring the effect of inhibiting the progression of joint destruction by MRA alone in early RA patients. It has been shown. In addition, by measuring the amount of sputum and MMP-3 after rheumatoid arthritis treatment (for example, at 12 weeks), the degree of progression of joint destruction can be predicted. In other words, it became clear that the prognosis of rheumatoid arthritis can be predicted. In addition, the above results show that the rate of change in sputum concentration in the early stage of treatment correlates with the amount of change in the JSN score during the longer treatment period, and the rate of change in MMP-3 concentration in the early stage of treatment is related to the erosion score of the longer treatment period. It shows that it correlates with the amount of change and the amount of change in JSN score. Because of this and these two biochemical markers showing changes independent of each other! /, According to the present invention, the measurement of soot amount can only be effective in predicting the prognosis of JSN. By combining JSN prognosis prediction by measurement and JSN and erosion prognosis prediction by measuring MMP-3, more accurate joint destruction prognosis can be predicted.

Industrial applicability

The present invention provides a method for predicting the prognosis of treatment for rheumatoid arthritis using the amount of PIIANP as an index in a subject who has undergone rheumatoid arthritis treatment.

According to the method of the present invention, it was possible to predict the prognosis of RA patients due to the treatment by examining the temporal change in the early treatment of rheumatoid arthritis.

In addition, by measuring changes in PIIANP levels over time, it has become possible to judge the effectiveness of therapeutic agents for rheumatoid arthritis and to select patients that are suitable for the treatment of rheumatoid arthritis.

Claims

The scope of the claims

[I] A method for predicting a prognosis for treatment of rheumatoid arthritis in a subject who has undergone rheumatoid arthritis treatment, comprising the following steps (a) to (c):

(a) Subject power Sample collection process

[2] The method according to claim 1, wherein, in the step (c) according to claim 1, when the amount of PIIANP is decreased as compared to before the start of treatment, the prognosis by the treatment is predicted to be good.

[3] In the early stage after the start of rheumatoid arthritis treatment! /, When the amount of PIIANP decreases compared to before the start of the treatment, the prognosis by the treatment is predicted to be good. The method of description.

[4] The method according to claim 2 or 3, wherein whether or not the prognosis of treatment for rheumatoid arthritis is favorable is determined by whether or not progression of joint destruction in rheumatoid arthritis is suppressed.

[5] The sample is collected from subjects over time, and the amount of PIIANP is measured.

The method according to 1.

6. The method according to any one of claims 1 to 5, wherein the rheumatoid arthritis is early rheumatoid arthritis.

7. The method according to claim 1, wherein the sample collected from the subject is plasma, serum, urine, or joint fluid.

[8] The method according to claim 1, wherein the rheumatoid arthritis treatment is treatment by administration of an anti-rheumatic agent.

[9] The rheumatoid arthritis treatment is treatment by administration of an IL-1 inhibitor, an IL-6 inhibitor, a TNF-a inhibitor, an IL-15 inhibitor, or an IL-17 inhibitor. The method described in 1.

[10] The method according to claim 1, wherein the rheumatoid arthritis treatment is treatment by administration of an antibody that inhibits IL-1, IL-6, TNF-a, IL-15 or IL-17 signaling.

[II] Rheumatoid arthritis treatment is treatment by administration of anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-TNF-a antibody, anti-CD20 antibody, methotrexate or CTLA4-IgG fusion protein. The method according to claim 1.

[12] A kit for predicting the prognosis of treatment in rheumatoid arthritis, comprising a reagent for measuring the amount of sputum.