WO2012067267A1 - Méthode d'essai faisant appel à un anticorps monoclonal anti-c1q - Google Patents

Méthode d'essai faisant appel à un anticorps monoclonal anti-c1q Download PDF

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Publication number
WO2012067267A1
WO2012067267A1 PCT/JP2011/077267 JP2011077267W WO2012067267A1 WO 2012067267 A1 WO2012067267 A1 WO 2012067267A1 JP 2011077267 W JP2011077267 W JP 2011077267W WO 2012067267 A1 WO2012067267 A1 WO 2012067267A1
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WIPO (PCT)
Prior art keywords
clq
patient
amount
protein
bone
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Application number
PCT/JP2011/077267
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English (en)
Inventor
Takahiro Ochi
Yasunori Shimaoka
Peter E. Lipsky
Original Assignee
Takahiro Ochi
Yasunori Shimaoka
Lipsky Peter E
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Filing date
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Application filed by Takahiro Ochi, Yasunori Shimaoka, Lipsky Peter E filed Critical Takahiro Ochi
Publication of WO2012067267A1 publication Critical patent/WO2012067267A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the present invention relates to a testing method using an anti-Clq monoclonal antibody and a testing kit therefor.
  • RA rheumatoid arthritis
  • Biological formulations approved for clinical use for RA so far include Infliximab (commercial name: Remicade) , Tocilizumab, Rituximab, Abatacept and the like.
  • Infliximab commercial name: Remicade
  • Tocilizumab Tocilizumab
  • Rituximab Rituximab
  • Abatacept Abatacept
  • One of the characteristics of these biological formulations is the high cost of treatment. Therefore, considering the financial burden on patients with RA as well as on the health system, it is important to decide whether or not it is necessary to use a biologic formulation in a specific RA patient.
  • Non-Patent Document 1 It has been reported that measurement of the amount of Clq protein in the blood from a patient with RA (Non-Patent Document 1) might be employed to identify patients with progressive joint damage . While an anti-Clq antibody has been developed by the present inventor (Patent Document 1) , a method for using this antibody to develop a prognosis in individual patients with RA concerning whether they will develop joint destruction and what is the nature of the joint destruction has not been established.
  • Patent Document 1 Ochi T et al., Arthritis Rheum. 1988 Jan; 31(1) : 37-43
  • Non-Patent Document 1 WO2008/035527 SUMMARY OF INVENTION
  • the problem to be solved by the present invention is to provide a testing method and a kit for determining whether an individual patient with RA will develop bone or joint destruction and what is the nature of the bone or joint destruction.
  • the present invention provides the followings :
  • a testing method for determining bone or joint destruction in an RA patient comprising the steps of:
  • step (B) the method according to (1) , which identifies in step (B) that the patient will develop bone or joint destruction of LES when the amount of Clq protein in the blood is 87 ⁇ g/ml or less; (3) the method according to (1) , which identifies in step (B) that the patient will develop bone or joint destruction of MES or MUD when the amount of Clq protein in the blood is 145 g/ml or more ;
  • the anti-Clq monoclonal antibody is capable of specifically recognizing a peptide having any amino acid sequence of SEQ ID NOs: 1 to 7;
  • the anti-Clq monoclonal antibody is an antibody produced by a hybridoma having an accession number selected from the group consisting of the accession numbers FERM BP- 10650, FERM BP- 10652, FERM BP- 10653 and FERM BP- 10654 from International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology;
  • bone or joint destruction due to RA can be determined in the early stage after disease onset, and consequently, active treatment of RA using a biological formulation or the like can be initiated earlier.
  • Figure 1 shows distributions of Clq protein in RA patients with minimal progressive joint destruction (LES) and patients with rapidly progressive destructive disease (MES/MUD) .
  • Figure 2 shows that the amount of Clq protein does not correlate with the amount of other marker proteins in patients with RA.
  • (A) shows correlation coefficients
  • (B) shows P-values.
  • the present invention relates to a testing method for determining bone or joint destruction using anti-Ciq monoclonal antibody (ies) .
  • the bone or joint destruction refers to that caused by RA.
  • RA patients with various degrees of bone or joint destruction are classified into least erosive subset (LES) that is relatively mild, and more erosive subset (MES) or mutilating disease (MUD) that is more severe based on morphological changes in radiographic images (roentgenograms) (see Ochi et al . , Prognosis of RA and surgical indications. Seikei-Geka, Vol.46, No.7, pp 880-888, 1995).
  • the nature of bone or joint destruction in a subject can be determined. For example, when an RA subject is determined to have more erosive or mutilating disease in the early stages after disease onset, more aggressive treatments, such as biological formulations, can be actively adopted.
  • the method of the present invention can be used for the prognosis of a subject who suffers from RA.
  • the present invention also provides anti-Clq monoclonal antibody (ies) used in the method mentioned above and the kit mentioned below.
  • a subject may be a human subject or may be a nonhuman subj ect .
  • the method of the present invention may comprise the steps of (A) measuring the amount of Clq protein in a sample obtained from an RA patient using at least one anti-Clq monoclonal antibody capable of specifically recognizing Clq protein, and (B) identifying that the patient will develop bone or joint destruction of the least erosive subset (LES) when the amount of Clq protein in the blood is 97 ⁇ g/ml or less, preferably 87 ⁇ g/ml to 97 ⁇ g/ml or less (e.g. , 95 ⁇ g/ml or less, or 90 or less) , and more preferably 87 ⁇ g/ml or less (e.g.
  • MES erosive subset
  • MOD mutilating disease
  • the anti-Clq monoclonal antibody used in the present invention can specifically bind to Clq protein.
  • Such an antibody is preferably one such that the amount of antigen-antibody conjugate to be formed increases depending on the amount of Clq protein, more specifically, one that can be used for the quantitative determination of Clq protein.
  • the anti-Clq monoclonal antibody used in the present invention may be obtained, for example, by immunizing a mammal such as a mouse with Clq protein, fusing lymphocytes of the immunized animal and a myeloma cell line, to produce a hybridoma, and culturing the hybridoma.
  • Such an antibody may be produced using other known methods, for example, a gene recombination method and a chemical synthetic method.
  • amino acid sequences of Clq protein epitopes and the nucleotide sequences encoding the amino acid sequences in a preferred aspect of the present invention are shown as SEQ ID NOs : 1 to 7 and SEQ ID NOs : 8 to 16, respectively.
  • the anti-Clq monoclonal antibody that may be used in the present invention is preferably one capable of specifically recognizing a peptide selected from the group consisting of (a) a peptide having any amino acid sequence of SEQ ID NOs: 1 to 7, (b) a peptide having an amino acid sequence having deletion, substitution or addition of one or more, preferably, one or several, for example, 2, 3, or 4 amino acids, in any one amino acid sequence of SEQ ID NOs: 1 to 7, and (c) a peptide having an amino acid sequence having a homology of at least 50% or more, for example, 70, 80, 90 or 93% or more, to any one amino acid sequence of SEQ ID NOs : 1 to 7.
  • the homology of the amino acid sequence can be determined using, for example, FASTA, BLAST and DNASIS (manufactured by Hitachi Software Engineering Co. , Ltd. ) , and GENETYX (manufactured by GENETYX CORPORATION) .
  • the anti-Clq monoclonal antibody used in the present invention may be obtained by immunizing an animal such as a mouse with the above-described peptide or a protein containing the peptide and fusing lymphocytes of the immune mouse and a myeloma cell line to produce a hybridoma.
  • the anti-Clq monoclonal antibody used in the present invention may be an anti-Clq monoclonal antibody produced by hybridoma KS-0131 #8, KS-0131 #33, KS-0131 #35, KS-0131 #40, KS-0131 #54 or KS-0131 #7, a fusion cell of a mouse myeloma cell line and a mouse lymphocyte.
  • hybridomas are deposited to the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki) and accepted on August 2, 2006, and the accession numbers FERM BP- 10649, FERM BP-10650 , FERM BP- 10651 , FERM BP-10652, FERM BP-10653, and FERM BP-10654 are given, respectively.
  • the anti-Clq monoclonal antibody used in the present invention may be prepared by culturing the above hybridoma under known culture conditions, for example, in a culture medium containing the following components: 425 mL of RPMI 1640 (Kohjin 16005005) , 5 mL of L-Glutamine (SIGMA G7513 ) , 5 mL of sodium pyruvate (SIGMA S8636) , 10 mL of HAT supplement (GIBCO 21060-017), 2.5 mL of penicillin-streptomycin (SIGMA P4333) , and 75 mL of FBS, at 37 ⁇ 0.1°C in 5+0.15% C0 2 , and collecting and purifying the produced anti-Clq monoclonal antibody by known procedures or methods.
  • anti-Clq monoclonal antibodies produced by hybridomas KS-0131 #8, KS-0131 #33, KS-0131 #35, KS-0131 #40, KS-0131 #54 and KS-0131 #76 may be represented as anti-Clq antibody #8, anti-Clq antibody #33, anti-Clq antibody #35, anti-Clq antibody #40, anti-Clq antibody #54 and anti-Clq antibody #76, respectively.
  • the anti-Clq monoclonal antibody used in the present invention includes the fragments thereof, and modified antibodies such as chimeric antibodies and humanized antibodies and mutated antibodies thereof. These fragments, modified antibodies or mutated antibodies also have specificity for Clq protein similar to the original antibody. These antibodies can be prepared by the procedure and method known to a skilled person in the art .
  • the anti-Clq monoclonal antibody used in the present invention one kind, or two or more kinds of the anti-Clq monoclonal antibodies may be used. By using two or more kinds of the anti-Clq monoclonal antibodies, determination sensitivity and specificity are increased, and the accuracy of determination by the present invention is improved.
  • the combinations of anti-Clq antibody #33 and anti-Clq antibody #54, anti-Clq antibody #33 and anti-Clq antibody #76, anti-Clq antibody #40 and anti-Clq antibody #54, and anti-Clq antibody #40 and anti-Clq antibody #76 are used in the method of the present invention.
  • the antibody used in the present invention may also be a labeled antibody.
  • Various labels are known and a person skilled in the art can properly select and use them.
  • enzymes such as horseradish peroxidase (HRP) , ALP, glucose oxidase, and ⁇ -galactosidase
  • fluorescent labels such as FITC, rhodamine, Cy3 , Cy5 , Texas Red, Alexa Fluors, BODIPYs, IRDyes, MFPs, Quantum Dots, AMCA, allophycocyanin, BMP, Cy2 , Cy3.5 , Cy5.5, DTAF, DyLight 547, DyLight 647, FluoroNanogold, phycoerythrin, phycocyanin, R-PE, saporin, and TRITC, chemical substances such as biotin, digoxigenin (DIG) , Acridium Ester, and Flashlight, beads such as 60 mm Microbead, magnetic beads such as MagCellect Ferrofluid
  • DIG digoxigenin
  • radiolabels such as 125 I
  • labels such as metal particles and agarose
  • the determination method of the present invention can be efficiently carried out by labeling the antibody.
  • the sample obtained from a subject used in the present invention may be any sample as long as it is possible to contain Clq protein, for example, blood, joint fluid, or the like, and blood is preferably used.
  • the step of determining the amount of Clq protein in a sample in the present invention may also be carried out, for example, by incubating the above sample with the above anti-Clq monoclonal antibody, forming a conjugate of the anti-Clq antibody and Clq protein in the sample, and subsequently determining the amount of the conjugate .
  • An ELISA microplate was bound to 100 ⁇ of 15 ⁇ 9/ ⁇ 1 anti-Clq monoclonal antibody (solid-phase antibody) solutions
  • a solution obtained by adding 1% BSA to a solution containing 0.05 M Tris, 0.05 glycine, 0.01 M EDTA and 0.1% NaN 3 at pH 8.0 was used as a diluent .
  • 100 ⁇ of a serum sample diluted with the above diluent by 2000-fold was added thereto, and the mixture was allowed to react at 37°C for 1 to 2 hours.
  • the reactant was washed twice, and thereafter, 100 ⁇ of a secondary antibody (anti-Clq monoclonal antibody adjusted to 15 ⁇ g/ml or anti-Clq polyclonal antibody) labeled with horseradish peroxidase (HRP) was added thereto, and the mixture was allowed to react at 37°C for 1 to 2 hours. Subsequently, the reactant was washed twice, and thereafter, 50 ⁇ of an Ortho-Phenylenedianmin (OPD) solution that is an HRP substrate solution was added thereto to react for 10 min. Thereafter, the reaction was stopped with 50 ⁇ of a stop solution (1 M sulfuric acid) . Finally, the absorbance of the reaction solution was determined at 490 nm.
  • OPD Ortho-Phenylenedianmin
  • the amounts of the other marker proteins in the blood are shown in Table 2.
  • the result of comparing the amounts of these marker proteins with the amount of Clq protein obtained in Example 1 is shown in Fig. 2 (In this figure, (A) shows correlation coefficients, and (B) shows P-values) .
  • the amount of Clq protein did not correlate with the other marker proteins (CRP, MMP-3, IL-6, anti-CCP-antibody, and the like) and was an independent unique value.
  • the amounts of the above marker proteins present in the blood largely change by administration of medications, such as glucocorticoids or the like.
  • the amount of Clq protein does not change and is constant for each patient with RA.
  • the method for determining the amount of Clq protein as a method to assess the degree of bone or joint destruction in patients with RA is a highly reliable and independent method that is not influenced by administration of glucocorticoids, or the like, and which is not interchangeable with other methods.
  • hand-sharp score (hand-SS) as an evaluation of joint destruction of the hand only was calculated. The method recommended by van der Heijde et al .
  • mTSS modified total sharp score
  • hand sharp score 21 examples of LES and 44 examples of MES/MUD were calculated.
  • the score values can vary in a range of 0 to 388, and in hand-SS, the score values, can vary in a range of 0 to 280.
  • the score values obtained in m-TSS and hand-SS are shown in Table 3. As seen from Table 3, significant differences in severity of bone and cartilage destruction were confirmed between LES and MES or MUD.
  • bone or joint destruction due to RA can be determined in the early stage after disease onset, and consequently, active treatment of RA using a biological formulation or the like can be initiated earlier.

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Abstract

Cette invention concerne une méthode d'essai permettant de déterminer la destruction de l'os ou de l'articulation chez un patient atteint de polyarthrite rhumatoïde à l'aide d'un anticorps monoclonal anti-C1q, et un kit d'essai afférent. Selon la présente invention, la destruction de l'os ou de l'articulation due à la polyarthrite rhumatoïde peut être déterminée à un stade précoce après apparition de la maladie.
PCT/JP2011/077267 2010-11-18 2011-11-18 Méthode d'essai faisant appel à un anticorps monoclonal anti-c1q WO2012067267A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10227398B2 (en) 2013-07-09 2019-03-12 Annexon, Inc. Anti-complement factor C1q antibodies and uses thereof
US10723788B2 (en) 2015-11-24 2020-07-28 Annexon, Inc. Anti-complement factor C1q Fab fragments and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008035527A1 (fr) * 2006-09-21 2008-03-27 Mmt Co., Ltd. ANTICORPS MONOCLONAL ANTI-C1q

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008035527A1 (fr) * 2006-09-21 2008-03-27 Mmt Co., Ltd. ANTICORPS MONOCLONAL ANTI-C1q

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YASUNORI SHIMAOKA: "Research on the Clq value as a severity-of-illness (type of disease) prognosis method of rheumatoid arthritis, The disease inducing factor elucidation and radical cure treatment research and development rheumatoid arthritis marrow blood", THE HEISEI 20 FISCAL YEAR GENERALIZATION / ASSIGNMENT RESEARCH REPORT, March 2009 (2009-03-01), pages 5 - 7 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10227398B2 (en) 2013-07-09 2019-03-12 Annexon, Inc. Anti-complement factor C1q antibodies and uses thereof
US10590190B2 (en) 2013-07-09 2020-03-17 Annexon, Inc. Anti-complement factor C1q antibodies and uses thereof
US10927167B2 (en) 2013-07-09 2021-02-23 Annexon, Inc. Anti-complement factor C1Q antibodies and uses thereof
US11649279B2 (en) 2013-07-09 2023-05-16 Annexon, Inc. Anti-complement factor C1Q antibodies and uses thereof
US10723788B2 (en) 2015-11-24 2020-07-28 Annexon, Inc. Anti-complement factor C1q Fab fragments and uses thereof
US11999779B2 (en) 2015-11-24 2024-06-04 Annexon, Inc. Anti-complement factor C1q Fab fragments and uses thereof

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