JP2006213737A - Bone resorption inhibitor including interleukin-6 receptor antibody as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new bone resorption inhibitor including interleukin-6 antibody as an active ingredient. <P>SOLUTION: The bone resorption inhibitor includes an antibody to interleukin-6 receptor as an active ingredient. This bone resorption inhibitor effectively inhibits the bone resorption for suppressing the formation of osteoclast cells in the presence of interleukin-6 and interleukin-6 receptor and is useful as a therapeutic agent for a variety of diseases relating to bone resorption. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a bone resorption inhibitor comprising an antibody against interleukin 6 receptor (hereinafter referred to as interleukin 6 receptor antibody) as an active ingredient.

Bone tissue is replaced with new bone without changing the basic shape by bone formation by osteoblasts and bone resorption by osteoclasts. This process is called bone remodeling (bone remodeling) and plays an important role in maintaining the functions of the living body.
However, once the balance between bone formation and bone resorption is lost, bone tissue becomes abnormal and exhibits various diseases.

Osteoclasts, which play a major role in bone resorption, are derived from macrophage cells and are formed by differentiating into osteoclasts through cell-cell contact with osteoblasts. It is called a bone resorption factor, and active vitamin D ₃ , parathyroid hormone, interleukin 1 (1L-1), prostaglandin, etc. are known so far.
It is known that excessive administration of these bone resorption factors increases and activates the number of osteoclasts in vivo and enhances bone resorption (see, for example, Medical History 165: 572-576, 1993) ).

  Interleukin 6 (IL-6) was discovered as a cytokine that promotes the proliferation of B lymphocyte cells and was subsequently found to affect the immune system by also inducing the maturation of T lymphocyte cells (eg, Lotz et al .; see J. Exp. Immunol. 18: 1253-1258, 1988). Furthermore, IL-6 is involved in various cell functions such as being involved in the induction of hematopoietic stem cell differentiation, and IL-6 is secreted from various cells, suggesting an effect on bone. ing.

  Ishimi et al. Reported that mouse osteoblasts produced large amounts of IL-6 by stimulation with bone resorption factor IL-1 and tumor necrosis factor (TNF), resulting in in vitro bone resorption. J. Immunol., 145: 3297-3803, 1990). IL-6 is also known to exhibit bone resorption activity on the mouse parietal bone (J. Immunol., 145: 3297-3803, 1990), and further, CHO cells transfected with the IL-6 gene are introduced into mice. When transplanted, it has been reported to exhibit hypercalcemia (Endocrinology, 128: 2657-2659, 1991).

  However, Al-humidan et al., Barton et al. Reported that bone resorption of the mouse parietal bone was not observed even when IL-6 was added (J Bone Miner Res 6: 3-8, 1991 and Cytokine 2: 217-220). , 1990), and Littlewood et al. Show that osteoblasts stimulated by parathyroid hormone (PTH), lipopolysaccharide (LPS), TNFα, or IL-1 produce IL-6, whereas IL-6 produces bone. Since it did not show any effect on the differentiation and proliferation of blast-like cells, it has been reported that IL-6 produced from osteoblasts does not affect the proliferation and differentiation of osteoblasts (J Bone Miner Res 6: 141-148, 1991).

Furthermore, Littlewood et al. Stated that IL-6 production of osteoblasts was increased by stimulation with PTH, but the amount of messenger RNA (mRNA) of IL-6 receptor (IL-6R) was not increased (Endocrinology, 129: 1513). -1520, 1991).
Therefore, the relationship between IL-6 and bone resorption has been reported to be contradictory depending on the materials used and the experimental system, and the relationship has not yet been elucidated (details are reviewed by Rodman: J Bone Miner Res 7: 475). -478, 1992).

  Recently, Manologas et al. Reported that estrogen suppresses IL-6 production in vitro (JCI 89: 883-891, 1992), or increased the number of osteoclasts in ovariectomized (OVX) rats. It has been reported that 6 antibodies suppress (Science 257: 88-91, 1992). However, in these reports, there is no data as to whether or not the increased osteoclasts are activated (mature cells) to the extent that they have bone resorption action, and therefore IL-6 is bone in vivo. It remains unclear whether it is directly involved in absorption.

The present inventors have intensively studied the role of IL-6 in bone resorption. However, in transgenic mice in which human IL-6 was forcibly expressed, hypercalcemia and increased osteoclast formation were not observed. As a result of studies on the contribution of factors other than IL-6, IL-6 alone has almost no osteoclast formation promoting action, but it has a strong osteoclast forming action in the presence of IL-6R. Furthermore, the present inventors have found that this osteoclast-forming action is suppressed by adding an anti-IL-6R antibody, thereby completing the invention.
That is, the present invention relates to a bone resorption inhibitor containing an interleukin 6 receptor antibody (IL-6R antibody) as an active ingredient.

As long as the anti-IL-6R antibody used in the present invention has an inhibitory effect on bone resorption, the origin of the antigen or antibody (difference in animal species) does not matter.
The anti-IL-6R antibody can be obtained as a polyclonal or monoclonal type antibody using known means. For example, in the case of an anti-human IL-6R polyclonal antibody, the gene sequence disclosed in European Patent Application Publication No. EP325474 is inserted into a known expression vector system and expressed in a suitable host cell. Alternatively, it can be obtained by purifying the target IL-6R protein from the culture supernatant and then immunizing mammals other than humans using the protein as a sensitizing antigen.

In the case of an anti-mouse IL-6R antibody, it can be obtained by using the same method as described above using the gene sequence disclosed in JP-A-3-15595.
On the other hand, there are two types of IL-6R: those expressed on the cell membrane and those released from the cell surface (hereinafter referred to as soluble), and soluble IL-6R is an IL-6R protein on the cell membrane. The structure is different in that the intracellular region of the extracellular region, the transmembrane region, and the intracellular region is missing.

Therefore, the antigen for the anti-IL-6R antibody of the present invention contains such soluble IL-6R, and these soluble IL-6R can be obtained by known means (for example, JP-A-4- 98800).
In the case of a monoclonal antibody, after immunizing a mammal using IL-6R protein as a sensitizing antigen, the plasma cells (immune cells) are fused with myeloma cells of mammals such as mice, and the resulting fused cells (hybridoma) Can be obtained by selecting a clone that neutralizes the action of IL-6R, culturing this, and recovering the desired antibody.

The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion, and is generally a mouse, rat, hamster. Etc. are used.
Mammalian myeloma cells as the other parent cells to be fused with the immune cells include various known cell lines such as P3 (P3X63Ag8.653) [J. Immunol., 123: 1548, 1978], p3-U1 [Current Topics in Microbiology and Immunology, 81: 1-7, 1978], NS-1 [Eur. J. Immunol., 6: 511-519, 1976], MPC-11 [Cell, 8: 405-415, 1976], SP2 / 0 [Nature, 276,269-270, 1978], FO [J. Immunol. Meth., 35: 1-21, 1980], S194 [J. Exp. Med., 148: 313. -323, 1978], R210 [Nature, 277: 131-133, 1979] etc. are preferably used.

The cell fusion between the immune cells and myeloma cells is basically performed according to a known method, for example, the method of Milstein et al. [Methods Enzymol., 73: 3-46, 1981]. It can be carried out.
More specifically, the cell fusion is performed in a normal nutrient medium in the presence of a fusion promoter, for example. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.

  For example, the usage ratio of immune cells and myeloma cells is preferably about 1 to 10 times that of myeloma cells. As the medium used for the cell fusion, for example, RPMI-1640 medium suitable for growth of the myeloma cell line, MEM medium, and other normal mediums used for this type of cell culture can be used. Serum supplements such as fetal calf serum (FCS) can be used in combination.

  In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the medium, and a PEG solution pre-warmed to about 37 ° C., for example, PEG having an average molecular weight of about 1,000 to 6,000, Usually, it is carried out by adding to a medium at a concentration of about 30 to 60% (W / V) and mixing. Subsequently, the target hybridoma is formed by sequentially adding an appropriate medium and centrifuging to remove the supernatant.

  The hybridoma is selected by culturing in a normal selection medium, for example, HAT medium (medium containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT medium is continued for a time sufficient for the cells other than the target hybridoma (unfused cells) to die, usually several days to several weeks. Subsequently, according to a normal limiting dilution method, screening of a hybridoma producing the target antibody and single cloning are performed.

  Furthermore, when the obtained antibody is an antibody derived from a non-human animal, the reconstituted human type in which the FR portion and the constant region portion are changed to a human-derived antibody while leaving the antigen recognition site (CDR). As an example of a human type antibody of IL-6R antibody, PCT International Publication No. WO 92/197559 can be mentioned.

  The anti-IL-6R antibody of the present invention can select an inter-species combination between IL-6R as an antigen and an antibody-producing cell depending on the animal species to be administered. In general, human-derived antibodies against human IL-6R are desirable for humans and mouse-derived antibodies against mouse IL-6R for mice, but different species are acceptable as long as they are clinically acceptable. A combination of the two may be used.

  As shown in Experiment 3 of Example 2, anti-IL-6R antibody, which is an active ingredient of the bone resorption inhibitor of the present invention, forms osteoclasts in the presence of mouse IL-6 and mouse soluble IL-6R. Suppress. Further, as shown in Example 3, anti-IL-6R antibody which is an active ingredient of the bone resorption inhibitor of the present invention is bone resorption in the presence of mouse IL-6, mouse soluble IL-6R and human IL-1α. Suppress.

The bone resorption inhibitor of the present invention is effective for the treatment of various bone metabolic diseases involved as long as bone resorption induced by IL-6R is suppressed. Examples of such diseases include osteoporosis, rheumatoid arthritis, multiple myeloma, neoplastic hypercalcemia, renal osteodystrophy, Paget's disease, bone metastasis, osteosarcoma and the like.
The bone resorption inhibitor of the present invention can be administered systemically or locally by conventional routes, for example, orally in the form of tablets or capsules or parenteral methods such as injections. Furthermore, it can take the form of a pharmaceutical composition or kit with at least one pharmaceutical carrier or diluent.

The dosage varies depending on the degree of disease state, administration method, and the like, and it is necessary to select an appropriate amount as appropriate. It is a dose. However, the bone resorption inhibitor of the present application is not limited to these doses.
It has not been reported that there is any influence on the toxicity of IL-6R antibody until now.

Hereinafter, the present invention will be specifically described with reference to Examples and Reference Examples.
Reference Example 1 Preparation of Mouse IL-6 About 10 ⁸ P388D ₁ (IL-1) cells (Nordan et al., Science 233: 566-569, 1986; Bazin et al., J. Immunol. 139: 780-787, 1987) Double-stranded CDNA was synthesized by random priming using Fast Track ^™ (Invitrogen) kit.

  Furthermore, as a primer, the translation initiation codon (34th ATG) of the mouse IL-6 gene (J. Van Snick et al., Eur. J. Immunol 18: 193, 1988) having a recognition site for the restriction enzyme BamH1 at the 5 'or 3' end. Oligomers complementary to the 21st to 43rd sequences including) and the 658th to 683rd sequences including the stop codon (the 667th tag) were synthesized.

  Using these primers and Gene Amp (Takara Shuzo) kit, 50 cycles of PCR were performed with DNA Thermo Cycler (Takara Shuzo) at 94 ° C for 1 minute, 50 ° C for 2 minutes, and 72 ° C for 3 minutes. The amplified fragment (0.66 kb) was purified by low-melting point agarose gel electrophoresis, treated with BamH1, introduced into the PUC19 vector, and subcloned. The mouse IL6 gene was excised with BamH1, and then incorporated into pdR previously treated with BamH1 to transduce CHO cells. A CHO cell resistant to 50 nM MTX was selected and the culture supernatant was used for the experiment.

Reference Example 2 Preparation of Mouse Soluble IL6 Receptor Antibody (1) Anti-mouse soluble IL-6 receptor antibody RS12 was obtained by the method of Saito et al. (J. Immunol., 147: 168-173 (1991)). This antibody was of the IgG2a subclass.

(2) CHO cells producing mouse soluble IL-6 receptor disclosed in the above-mentioned literature such as Saito are cultured in IMDM medium containing 10% FCS, and the culture supernatant is RS12 antibody and Affigel 10 gel (BioRad). It refine | purified using the affinity column fixed to. 50 μg of the obtained mouse soluble IL-6 receptor was mixed with Freund's complete adjuvant and inoculated subcutaneously into the abdomen of Wistar rats (Charles River Japan). Two weeks later, booster immunization was performed with Freund's incomplete adjuvant. On day 45, about 2 × 10 ⁸ spleen cells were fused with 1 × 10 ⁷ mouse myeloma cells P3U1 and 50% PEG1500 (Boehringer Mannheim) in a conventional manner, and then HAT medium was used. Hybridomas were selected.

  Hybridoma culture supernatant was added to an immunoplate coated with a rabbit anti-rat IgG antibody (cappel), and then mouse soluble IL-6R was reacted, followed by a rabbit anti-mouse IL-6R antibody and an alkaline phosphatase-labeled sheep anti-rabbit IgG antibody The hybridoma producing an antibody against the mouse soluble IL-6 receptor was screened by the ELISA method described above. Those in which the production of the antibody was confirmed were subcloned twice to obtain a single clone (MR16-1).

The neutralizing activity of the antibody produced by this hybridoma on mouse IL-6 was determined as MH60. It was examined by ³ H-thymidine incorporation using BSF2 cells (Matsuda et al., Eur. J. Immunol. 18: 951-956, 1988).
MH60. BSF2 cells were prepared to 1 × 10 ⁴ cells / 200 μl / well, mouse IL-6 (10 pg / ml), MR16-1 antibody and 12.12-1000 ng / ml of RS12 antibody were added thereto, and the mixture was incubated at 37 ° C. After culturing at 5% CO ₂ for 44 hours, ³ H-thymidine (IμCi / well) was added, and the uptake after 4 hours was measured (FIG. 1).

Example 1
Eight-week-old female ddy mice were subjected to sham surgery (sham) or ovariectomy (OVX) and sacrificed two weeks later. Bone marrow was collected from the tibia and femur using phenol red-free αMEM medium, and the bone marrow cells were removed by centrifugation to obtain bone marrow fluid. The bone resorption activity of the bone marrow fluid was measured by an organ culture method using mouse long bones labeled with ⁴⁵ Ca. That is, ⁴⁵ CaCl ₂ was subcutaneously administered to a mother mouse, and ⁴⁵ Ca-labeled forearm bones were collected from a 17-day-old fetus and organ-cultured in phenol red-free αMEM medium at 5% CO ₂ and 37 ° C. did.

After 24 hours, the bone marrow sample was added at a rate of 40% at the same time as replacing with phenol red-free αMEM medium containing 0.2% BSA. Bone resorption activity was approximately 25% in the Sham group, compared to approximately 60% in the OVX group.
Therefore, using this system, the bone marrow sample was pretreated (37 ° C., 2 hours, 5% CO ₂ ) with 66 μg / ml mouse IL-6 antibody and RS12 antibody, and as a result, Both antibodies inhibited bone resorption activity in the OVX group (FIG. 2).

Example 2
The number of osteoclasts formed by the co-culture system of mouse osteoblasts and bone marrow cells (Takahashi, et al .: Endocrinology 122: 1373, 1988, Takahashi, et al .: Endocrinology 123: 2600, 1988) was used as an index of bone resorption. Using.

  Mouse osteoblasts were prepared by the following method. That is, a skull removed aseptically from a 1-day or 2-day-old ddy mouse is placed in PBS containing 0.1% collagenase (for cell dispersion, Wako Pure Chemical Industries) and 0.2% dispase (joint spirit) 37 ° C. And shaken for 10 minutes. The floating cells were collected, and a new enzyme solution was added and the enzyme treatment was performed for 10 minutes. This enzyme digestion was repeated 5 times, and the cells that had floated in the 2nd to 5th digestion were collected as osteoblasts.

  Mouse bone marrow cells were prepared by the following method. That is, the tibia was aseptically removed from 6-9 week old ddy mice, and the bone ends were cut off. 1 ml of α-minimum essential medium (α-MEM, GIBCO) was injected from a distal end of the tibia with a syringe with a 25 G needle, and bone marrow cells were collected from the proximal end. Both of these cells were suspended in α-MEM containing 10% fetal bovine serum (Biocell) and used for culture.

In the co-culture, 1 × 10 ⁴ cells / 0.5 m / well osteoblasts and 2 × 10 ⁵ cells / well bone marrow cells are added to a 48-well culture plate and incubated at 37 ° C. with 5% CO ₂ for 6 to 6 times. Cultured for 7 days. The formed osteoclasts were identified by staining with tartrate-resistant acid phosphatase (TRAP), a marker enzyme for osteoclasts [Takahashi, et al .: Endocrinology 122: 1373, 1988].

  These culture systems include mouse IL-6 or mouse soluble IL-6R [IL-6sR (sR324)] [Saito, et al. J Immunol, 147: 168-173 (1991)] and mouse IL-6R antibody (MR16- 1 or RS12) or mouse IL-6 antibody (R & D systems) was added and the following experiment was performed. In addition, mouse IL-6 and sR324 used the culture supernatant of the cell which integrated these genes in the CHO cell, respectively. The concentrations of mouse IL-6 and IL-6sR324 in the culture supernatant were measured by enzyme immunoassay, diluted to an appropriate concentration with α-MEM, and used for the experiment.

Experiment 1 Osteoclast formation experiment with mouse IL-6 or mouse soluble IL-6R alone Break with mouse IL-6 (0.2 ng / ml to 200 ng / ml) and sR324 (0.5 to 500 ng / ml) alone Bone cell formation ability was examined, but none of them showed osteoclast formation ability alone. The active vitamin D ₃ is a positive control drug in this experimental system showed significant osteoclast formation (Figure 3).

Experiment 2 Osteoclast formation experiment in the presence of mouse IL-6 and mouse soluble IL-6R When 0.05 ng / ml to 500 ng / ml of sR324 was allowed to coexist in 20 ng / ml or 200 ng / ml of mouse IL-6 When the osteoclast-forming ability was examined, the formation of marked osteoclasts was observed depending on the concentration of sR324 (FIG. 4). When the formed osteoclasts were cultured on ivory slices, many resorption pits were observed, which were suppressed by the addition of calcitonin. Therefore, the osteoclasts formed in this experimental system were considered to be mature osteoclasts with bone resorbability and calcitonin receptor.

Experiment 3 Inhibitory effect of mouse anti-IL-6 receptor antibody (MR16-1 or RS12) or mouse anti-IL-6 antibody on osteoclast formation in the presence of mouse IL-6 and mouse soluble IL-6 receptor 20 ng / ml or The ability of osteoclasts to form when 62.5 ng / ml to 500 ng / ml of sR324 coexisted with 200 ng / ml of mouse IL-6 was markedly dependent on the concentration of sR324 as in Experiment 2. Osteoclast formation was observed. MR16-1 exhibited a concentration-dependent inhibitory effect on osteoclast formation by 20 ng / ml mouse IL-6 and 500 ng / ml sR324 in a concentration range of 1 ng / ml to 100 ng / ml. RS12 and mouse anti-IL-6 antibody showed inhibitory action at 10 ng / ml and 100 ng / ml (FIG. 5).

Example 3 Inhibitory effect of anti-mouse IL-6 receptor antibody (MR16-1) on bone resorption induced in the presence of mouse IL-6, mouse soluble IL-6 receptor and human IL-1α ⁴⁵ CaCl ₂ the the ⁴⁵ Ca labeled calvaria taken from fetuses of gestational 16 days old by subcutaneous administration, were organ cultured at 5% CO _2, 37 ℃ in BGJb medium containing BSA of 1 mg / ml. After 24 hours, the medium was replaced with fresh medium, and 100 pg / ml human IL-1α, 20 ng / ml mouse IL-6 and 500 ng / ml mouse soluble IL-6R (sR324) were added, and the mixture was incubated at 37 ° C., 5 Incubated with% CO ₂ for 5 days. In this case, MR16-1 as a test substance was added to the new medium at 10 μg / ml. Bone resorption induced by this experimental system was almost completely suppressed by 10 μg / ml MR16-1 (FIG. 6).

As described above, the bone resorption inhibitor of the present invention comprising an anti-IL-6R antibody as an active ingredient suppresses the formation of osteoclasts in the presence of IL-6 and IL-6R, and inhibits bone resorption. Proven to have an effect. Therefore, the bone resorption inhibitor of the present invention is used for various bone metabolic diseases in which bone resorption is involved, such as osteoporosis, rheumatoid arthritis, multiple myeloma, neoplastic hypercalcemia, renal osteodystrophy, pagetosis, bone Expected to be a therapeutic agent for metastasis and osteosarcoma.

1 shows MH60. It is a graph which shows the effect of antibody MR16-1 and RS12 with respect to IL-6 dependent proliferation of a BSF2 cell. FIG. 2 is a graph showing the neutralizing effect of antibodies on bone resorption activity of bone marrow fluid from OVX (2 weeks) mice. FIG. 3 is a graph showing the effect of IL-6 or soluble IL-6R (sR324) alone on the formation of osteoclasts in a co-culture system of mouse osteoblasts and bone marrow cells. FIG. 4 is a graph showing the effect of combined use of IL-6 or sR324 on the formation of osteoclasts in a co-culture system of mouse osteoblasts and bone marrow cells. FIG. 5 is a graph showing the inhibitory effect of IL-6R antibody on the formation of osteoclasts by coculture of mouse osteoblasts and bone marrow cells in the presence of IL-6 and IL-6R (sR324). FIG. 6 is a graph showing the inhibitory effect of anti-IL-6R antibody on ⁴⁵ Ca release in the presence of IL-6, soluble IL-6R (sR324) and IL-1α.

Claims (3)

  A bone resorption inhibitor comprising an antibody against interleukin-6 receptor as an active ingredient.   The bone resorption inhibitor according to claim 1, wherein the antibody against the interleukin 6 receptor is derived from a mouse.   The bone resorption inhibitor according to claim 1 or 2, wherein the interleukin 6 receptor is derived from a mouse.

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