WO2012067267A1

WO2012067267A1 - TESTING METHOD USING ANTI-C1q MONOCLONAL ANTIBODY

Info

Publication number: WO2012067267A1
Application number: PCT/JP2011/077267
Authority: WO
Inventors: Takahiro Ochi; Yasunori Shimaoka; Peter E. Lipsky
Original assignee: Takahiro Ochi; Yasunori Shimaoka; Lipsky Peter E
Priority date: 2010-11-18
Filing date: 2011-11-18
Publication date: 2012-05-24

Abstract

The present invention is directed to a testing method for determining bone or joint destruction in a patient with rheumatoid arthritis using an anti-C1q monoclonal antibody, and a test kit therefor. According to the present invention, bone or joint destruction due to rheumatoid arthritis can be determined in the early stage after disease onset.

Description

DESCRIPTION

TITLE OF INVENTION

TESTING METHOD USING ANTI-Clq MONOCLONAL ANTIBODY

TECHNICAL FIELD

[0001]

The present invention relates to a testing method using an anti-Clq monoclonal antibody and a testing kit therefor.

RELATED CASE(S)

This application claims the priority of Japanese Patent Application No. 2010-257843, filed November 18, 2010, the disclosures of which are incorporated herein by reference in their entirety.

BACKGROUND ART

[0002]

In recent years, the role of biological formulations has been increasing in the treatment of patients with rheumatoid arthritis (RA) , because of their excellent clinical effects. Biological formulations approved for clinical use for RA so far include Infliximab (commercial name: Remicade) , Tocilizumab, Rituximab, Abatacept and the like. 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.

[0003]

In the treatment of RA, it is recommended to detect RA as early as possible, and to start active treatment. As a result of such a treatment, the long term consequences of RA can be mitigated and, insome examples, the treatment may induce remission in disease activity. Patients with RA can be classified into a group with aggressive disease associated withthe development of severe joint destruction and a group of with less severe disease. It is essential to recognize the nature of RA in individual patients early in the course of disease so that appropriate therapy can be initiated. However, this is difficult to accomplish at the early stages of the disease. Although a number of biomarkers have been applied, none has proven to be successful in discriminating subjects with destructive versus milder disease early in the course of RA.

[0004]

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.

BACKGROUND ART DOCUMENTS PATENT DOCUMENT

[0005]

Patent Document 1: Ochi T et al., Arthritis Rheum. 1988 Jan; 31(1) : 37-43

NON-PATENT DOCUMENT

[0006]

Non-Patent Document 1: WO2008/035527 SUMMARY OF INVENTION

PROBLEMS TO BE SOLVED BY INVENTION

[0007]

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.

MEANS FOR SOLVING PROBLEMS

[0008] As a result of intensive studies in view of the above situation, the present inventors have succeeded in determining the prognosis that an RA patient will develop bone or joint destruction by measuring the amount of Clq protein in the blood using anti-Clq monoclonal antibodies developed by the present inventor (see WO2008/035527) , and thus have completed the present invention.

[0009]

More specifically, the present invention provides the followings :

(1) a testing method for determining bone or joint destruction in an RA patient, comprising the steps of:

(A) measuring the amount of Clq protein in the blood obtained from the 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 μg/ml or less, and identifying that the patient will develop bone or , joint destruction of the more erosive subset (MES) or the subset with mutilating disease (MUD) when the amount of Clq protein in the blood is 133 μg/ml or more;

(2) 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 μg/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 ;

(4) the method according to any one of (1) to (3) for prognostication of an individual subject who suffers from RA;

(5) the method according to any one of (1) to (4) , wherein the anti-Clq monoclonal antibody is capable of specifically recognizing a peptide having any amino acid sequence of SEQ ID NOs: 1 to 7;

(6) the method according to (5) , wherein 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;

(7) the method according to any one of (1) to (6) , wherein at least two anti-Clq monoclonal antibodies are used; and

(8) the method according to any one of (1) to (7) , wherein the amount of Clq protein is measured by ELISA method.

(9) a testing kit used in the method according to any one of (1) to (8) for determining bone or joint destruction in an RA patient, which comprises a Clq antibody as an essential component . EFFECTS OF INVENTION

[0010]

^"According to the present invention, 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.

BRIEF DESCRIPTION OF DRAWINGS

[0011]

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. In this figure, (A) shows correlation coefficients, and (B) shows P-values.

MODE FOR CARRYING OUT INVENTION

[0012]

In one aspect, the present invention relates to a testing method for determining bone or joint destruction using anti-Ciq monoclonal antibody (ies) . In the present invention, the bone or joint destruction refers to that caused by RA. In addition, 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). According to the method of the present invention, 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. In addition, by the above characteristics, 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.

[0013]

In the present invention, a subject may be a human subject or may be a nonhuman subj ect .

[0014]

In order to determine bone or joint destruction, 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 μg/ml or less, preferably 87 μg/ml to 97 μg/ml or less (e.g. , 95 μg/ml or less, or 90

or less) , and more preferably 87 μg/ml or less (e.g. , 85 μg/ml or less) , and identifying that the patient will develop bone or joint destruction of the more erosive subset (MES) or mutilating disease (MUD) when the amount of Clq protein in the blood is 120 μg/ml to 133 μg/ml or more, preferably 133 μg/ml to 145 μg/ml or more (e.g. 135 μg/ml or more, or 140 μg/ml or more), and more preferably 145 μg/ml or more.

[0015]

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. [0016]

The 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. More specifically, 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.

[0017] In a more preferred aspect, 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. These 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% C0₂ , and collecting and purifying the produced anti-Clq monoclonal antibody by known procedures or methods. Herein, the 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.

[0018]

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 .

[0019]

As to 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. Preferably, 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. For example, enzymes such as horseradish peroxidase (HRP) , ALP, glucose oxidase, and β-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

(registered trademark) , radiolabels such as ¹²⁵I, and labels such as metal particles and agarose may be used. The determination method of the present invention can be efficiently carried out by labeling the antibody.

[0020]

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.

[0021]

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 . The present invention may also be carried out using, for example, ELISA such as sandwich ELISA, immunoblotting, radioiramuno assay (RIA) , immunoprecipitation, a method using a protein array, a method using a flow cytometer, chemiluminescent enzyme immunoassay (CLEIA) , bioluminescent enzyme immunoassay (BLEIA) , a determination method using a developing material capable of transporting a solution by capillary action (test strip for immunoassay) , immunochromatography, immunostaining, agglutination, or the like. ELISA is preferable since special and expensive equipments or the like are not required in ELISA. Further, the present invention provides a testing kit used in the method mentioned above for determining bone or joint destruction in an RA patient, which comprises as an essential component a Clq antibody, preferably that deposited as mentioned above. Usually, an instruction for use is attached to the kit.

[0022]

The present invention will be hereinafter described in detail and specifically with reference to examples, however, the present invention is not limited to these examples.

EXAMPLE 1

[0023]

Distributions of Clq Protein in Patients with Mild Erosive Disease RA (LES) and Patients with Severe Erosive Disease (MES/MUD) For 73 examples (26 examples of LES, and '73 examples of MES/MUD) of patients with RA whose disease types were evident, the amounts of serum Clq protein were determined using an anti-Clq monoclonal antibody, and the distributions were studied.

[0024]

Quantitative Determination of Serum Clq Protein by Sandwich ELISA

An ELISA microplate was bound to 100 μΐ of 15 μ9/Ίη1 anti-Clq monoclonal antibody (solid-phase antibody) solutions For the preparation of the above solution, 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% NaN₃ at pH 8.0 was used as a diluent . Next, 100 μΐ 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 μΐ of a secondary antibody (anti-Clq monoclonal antibody adjusted to 15 μg/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 μΐι 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 μΏ of a stop solution (1 M sulfuric acid) . Finally, the absorbance of the reaction solution was determined at 490 nm.

[0025]

The results are shown in Table 1 and Figure 1. It was revealed from these results that the amount of Clq protein contained in the serum of RA patients with MES or MUD was significantly high as compared to that of RA patients with LES.Data shown are the mean+/-standard deviation.

[0026] ^

[Table 1]

Table 1: Amounts of Clq Protein in Each Subset of RA (MeaniSD)

EXAMPLE 2

[0027]

Correlation Between Amount of Clq Protein and Amounts of Other Marker Proteins

The presence or absence of a correlation between the amount of Clq protein and the amounts of other marker proteins contained in the blood was determined.

[0028]

For all 73 examples, including those from (LES) and MES/MUD, determinations of other marker proteins considered as clinically useful were carried out simultaneously (with the same samples) , together with that of serum Clq protein by sandwich ELISA. Levels of the other marker proteins, CRP, ESR (erythrocyte sedimentation) , RF (rheumatoid factor) , Anti-CCP Ab (anti-CCP antibody) , IL-6 (interleukin 6) and MMP-3 (matrix metal proteinase 3) were determined. The determinations were carried out using a method which had already been established in clinical use (a routine method) .

[0029]

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) . As seen from Figure 2, 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. It is known that the amounts of the above marker proteins present in the blood largely change by administration of medications, such as glucocorticoids or the like. On the other hand, it is known that the amount of Clq protein does not change and is constant for each patient with RA. Accordingly, it was revealed that the method for determining the amount of Clq protein as a method to assess the degree of bone or joint destruction in patients with RAis 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.

[0030]

[Table 2]

Table 2 : Amount of Each Marker Protein in Patients with Rheumatoid Arthrit (Mean±SD)

EXAMPLE 3

[0031]

Correlation Between the Amount of Clq Protein in blood and Bone Destruction

(1) Confirmation of Bone Destruction in Each Disease Subset (LES, MES and MUD)

It was confirmed that the severity of bone destruction clearly varied among disease types of RA (LES, and MES or MUD) , by using modified total Sharp score (m-TSS) , an indication of bone and cartilage destruction on radiograph. In addition, each disease type was also evaluated by using the Ssharp score method. It should be noted that the Sharp Score only evaluates joint damage in the hand (hand-SS) .

[0032]

In order to obtain indications of bone destruction in an individual patient in the same period as the blood samples of the patient with RA were collected, 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 .

(Lancet. 1989 May 13; 1(8646): 1036-8.) that includes evaluation of both hand and foot radiographs was alsoemployed As for modified total sharp score (mTSS) , 15 examples of LES and 40 examples of MES/MUD were calculated. As for hand sharp score (hand-SS) , 21 examples of LES and 44 examples of MES/MUD were calculated.

[0033]

In mTSS, 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. In both m-TSS and hand-SS, it is shown that the higher the score value, the higher the severity of bone and cartilage destruction. 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.

[0034]

[Table 3]

Table 3: Differences in Bone Destruction Between LES and MES/ UD

[0035]

(2) Correlation Between Amount of Clq Protein in blood and Bone and cartilage Destruction

The correlation between the evaluations of bone and cartilage destruction by m-TSS and hand-SS obtained in Example 3 (1) and the amount of Clq protein in the blood was studied.

[0036]

For RA patients LES and severe disease type (MES/MUD (55 examples of patients whose mTSS could be calculated, 65 examples of patients whose hand-SS could be calculated) , the correlations of serum Clq protein by sandwich ELISA with mTSS and hand-SS were studied according to a routine method.

[0037]

The correlation coefficients are shown in Table 4. As shownfrom Table 4, the amount of Clq protein in the blood exhibited a correlation coefficient in a range of about 0.28 to 0.31 with the evaluation of bone and cartilage destruction by sharp score, and significant correlation was shown. It was confirmed from this result that the disease subset of RA can be identified by measuring the amount of Clq protein in the blood using an anti -Clq monoclonal antibody. [0038]

[Table 4]

Table 4 : Correlations Between Amount of Clq Protein and

Bone Destruction

INDUSTRIAL APPLICABILITY

[0039]

According to the present invention, 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.

Claims

1. A testing method for determining bone or joint destruction in a patient with rheumatoid arthritis, comprising the steps of :

(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 μg/ml or less, and identifying that the patient will develop bone or joint destruction of the more erosive subset (MES) or the subset with mutilating disease (MUD) when the amount of Clq protein in the blood is 133 g/ml or more.

2. The method according to claim 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 μg/ml or less .

3. The method according to claim 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 μg/ml or more.

4. The method according to any one of claims 1 to 3 for prognostication of a subject who suffers from RA.

5. The method according to any one of claims 1 to 4 , wherein the anti-Clq monoclonal antibody is capable of specifically recognizing a peptide having any amino acid sequence of SEQ ID NOs : 1 to 7.

6. The method according to claim 5 , wherein 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.

7. The method according to any one of claims 1 to 6 , wherein at least two anti-Clq monoclonal antibodies are used.

8. The method according to any one of claims 1 to 7 , wherein the amount of Clq protein is measured by ELISA method.

9. A testing kit used in the method according to any one of claims 1 to 8 for determining bone or joint destruction in a patient with rheumatoid arthritis , which comprises a Clq antibody as an essential component .