JPH07165608A - Bone absorption inhibitor comprising interleukin 6 receptor antibody as active ingredient - Google Patents

Abstract

PURPOSE:To obtain a bone absorption inhibitor effective as a therapeutic agent for diseases related to bone absorption, capable of suppressing formation of osteoclast in the presence of interleukin 6 (IL-6) and interleukin 6 receptor (IL-6R). CONSTITUTION:This bone absorption inhibitor comprises an antibody against IL-6R as an active ingredient. IL-6 hardly shows promotive action on osteoclast formation by itself, but has a strong osteoclast formation promotion action in the presence of IL-6R. The osteoclast formation promotion action is checked by addition of anti-IL-6 antibody. IL-6R has two kinds whose one develops on a cell membrane and the other of which is released from a cell surface layer (soluble IL-6R). The soluble IL-6R is structurally different with respect to deficiency in intracellular region among an extracellular region, a cell penetration region and the intracellular region in IL-R on the cell membrane. The soluble IL-6R is used as an antigen to prepare anti-IL-6 antibody.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a bone resorption inhibitor containing an antibody against interleukin 6 receptor (hereinafter referred to as interleukin 6 receptor antibody) as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Bone tissue is replaced with new bone without changing its basic shape due to bone formation by osteoblasts and bone resorption by osteoclasts. This process is called bone remodeling,
It plays an important role in maintaining the function of the living body. However, once the balance between bone formation and bone resorption is lost, bone tissue becomes abnormal and various diseases are exhibited.

Osteoclasts, which play a major role in bone resorption, are derived from macrophage cells and are formed by differentiation into osteoclasts through intercellular contact with osteoblasts. This process is promoted. The factor that does this is called bone resorption factor, and active vitamin D ₃ , parathyroid hormone, interleukin 1 (1L-1), prostaglandin, etc. have been known so far. Excessive administration of these bone resorption factors causes i
It is known to increase and activate the number of osteoclasts and enhance bone resorption in n vivo (for example, medical history 16
5: 572-576, 1993).

Interleukin 6 (IL-6) was discovered as a cytokine that promotes the proliferation of B lymphoid cells and was subsequently found to affect the immune system by also inducing maturation of T lymphoid cells. (See, for example, Lotz et al .; 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, which suggests an effect on bone. ing.

In Ishimi et al., Mouse osteoblasts produce a large amount of IL-6 by stimulation with bone resorption factor IL-1 and tumor necrosis factor (TNF), and as a result, in vitro.
It was reported that bone resorption was induced in (J. Immunol., 14
5: 3297-3803, 1990). It is also known that IL-6 exhibits bone resorption activity on mouse parietal bone (J. Immuno
l., 145: 3297-3803, 1990), and when CHO cells transfected with the IL-6 gene are transplanted into mice, it is reported that they exhibit hypercalcemia (Endocrinology, 128: 265.
7-2659, 1991).

However, Al-humidan or B
Arton et al. reported that bone resorption of mouse parietal bone was not observed even when IL-6 was added (J Bone M
inerRes 6: 3-8, 1991 and Cytokine 2: 217-220, 199
0), and Littlewood et al., Include parathyroid hormone (PTH), lipopolysaccharide (LPS), T
Osteoblasts stimulated by NFα or IL-1 produce IL-6, but IL-6 showed no effect on the differentiation and proliferation of osteoblast-like cells. It has been reported that IL-6 produced by Escherichia coli does not affect the proliferation and differentiation of osteoblasts (J Bone Miner Res
6: 141-148, 1991).

Furthermore, although Littlewood et al. Increased IL-6 production in osteoblasts by PTH stimulation, I
Messenger RN of L-6 receptor (IL-6R)
It states that the amount of A (mRNA) did not increase (Endo
crinology, 129: 1513-1520, 1991). Therefore, IL-6
Regarding the relationship between bone resorption and bone resorption, contradictory results have been reported depending on the materials used and experimental systems, and the relationship has not yet been elucidated (for details, see Rodman's review: J Bone M
iner Res 7: 475-478, 1992).

Recently, Manolagas et al.
In vitro, estrogen suppresses IL-6 production (JCI 89: 883-891, 1992) or ovariectomy (O
VX) increased the number of osteoclasts in rats by estrogen and anti-I
Inhibition by L-6 antibody (Science 257: 88-91, 1992)
Is reported. However, in these reports, there is no data on whether or not the increased osteoclasts are activated (mature cells) to the extent that they have a bone resorption effect. It is still unclear if it is directly involved in absorption.

[0009]

Means for Solving the Problems The present inventors have diligently studied the role of IL-6 in bone resorption.
High Ca in transgenic mice forcibly expressing
Since no increase in blood plasma or osteoclast formation was observed, the results of studies on contributions of factors other than IL-6 showed that
IL-6 alone exhibits almost no osteoclastogenesis-promoting action, whereas IL-6R has a strong osteoclastogenesis action, and further, this osteoclastogenesis action is anti-I.
They found that it was suppressed by adding the L-6R antibody, and completed 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.

[0010]

[Detailed Description] The anti-IL-6R antibody used in the present invention may be derived from antigens or antibodies (different animal species) as long as it has a bone resorption inhibitory effect. The anti-IL-6R antibody can be obtained as a polyclonal or monoclonal type antibody using known means. For example, anti-human I
In the case of the L-6R polyclonal antibody, the gene sequence disclosed in European Patent Application Publication No. EP 325474 is inserted into a known expression vector system and expressed in a suitable host cell, and then in the host cell or in culture. It can be obtained by purifying the target IL-6R protein from the serum and then immunizing a mammal other than human with the protein as a sensitizing antigen.

In the case of anti-mouse IL-6R antibody, it can be obtained by using the gene sequence disclosed in Japanese Patent Laid-Open No. 3-155795 and using the same method as above. On the other hand, there are two types of IL-6R, one that is expressed on the cell membrane and one that is released from the cell surface (hereinafter referred to as soluble). Soluble IL-6R is IL-6 on the cell membrane.
The R protein is structurally different in that the intracellular region among the extracellular region, the cell transmembrane region, and the intracellular region is deleted.

Therefore, the antigen for the anti-IL-6R antibody of the present invention includes such soluble IL-6R, and these soluble IL-6R can be obtained by a known means (for example, a special method). See Kaihei 4-98800). In the case of a monoclonal antibody, the IL-6R protein is used as a sensitizing antigen to immunize a mammal, and then its plasma cells (immune cells) are fused with myeloma cells of a mammal such as a mouse to obtain a fused cell (hybridoma). It is possible to obtain the desired antibody by cloning it, selecting a clone that neutralizes the action of IL-6R from the clone, culturing the clone, and recovering the desired antibody.

The mammal to be immunized with the sensitizing antigen is not particularly limited, but it is preferable to select it in consideration of compatibility with myeloma cells used for cell fusion, and in general, mouse, Rats, hamsters, etc. are used. As a mammalian myeloma cell as the other parent cell to be fused with the above-mentioned immune cell, various known cell lines such as P3 (P3X63Ag8.653) are known.
[J. Immunol., 123: 1548, 1978], p3-U1 [Curren
t Topics in Micro-biology and Immunology, 81: 1-7,
1978], NS-1 [Eur. J. Immunol., 6: 511-519, 197].
6], MPC-11 [Cell, 8: 405-415, 1976], SP
2/0 [Nature, 276, 269-270, 1978], FO [J. Imm
unol. Meth., 35: 1-21, 1980], S194 [J. Exp. Me
d., 148: 313-323, 1978], R210 [Nature, 277: 131-
133, 1979] and the like are preferably used.

The cell fusion between the immune cell and the myeloma cell is basically a known method, for example, the method of Milstein et al. [Method]
S Enzymol., 73: 3-46, 1981] and the like. More specifically, the cell fusion is carried out in an ordinary nutrient medium in the presence of a fusion promoter, for example. Examples of the fusion accelerator include polyethylene glycol (PE
G), Sendai virus (HVJ) and the like are used, and if desired, an auxiliary agent such as dimethyl sulfoxide can be added and used in order to enhance the fusion efficiency.

The ratio of the immune cells to the myeloma cells used is, for example, 1 to 1 for the myeloma cells.
It is preferably about 10 times. Examples of the medium used for cell fusion include RPMI-1640 medium suitable for growth of the myeloma cell line, MEM medium, and the like,
An ordinary medium used for this type of cell culture can be used, and a serum supplement such as fetal calf serum (FCS) can also be used in combination.

In the cell fusion, a predetermined amount of the immune cells and myeloma cells are well mixed in the medium and pre-heated to about 37 ° C. in a PEG solution, for example, an average molecular weight of 100
About 0-6,000 PEG is usually added to the medium at about 30-
It is performed by adding at a concentration of 60% (W / V) and mixing. Subsequently, the desired hybridoma is formed by repeating the operation of successively adding an appropriate medium and centrifuging to remove the supernatant.

The hybridoma is a conventional selection medium,
For example, it is selected by culturing in HAT medium (medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium is continued for a time sufficient for killing cells (unfused cells) other than the target hybridoma, usually for several days to several weeks. Then, according to a usual limiting dilution method, screening and monocloning of hybridomas producing the desired antibody are carried out.

Furthermore, when the obtained antibody is derived from a non-human animal, its antigen recognition site (CD
Reconstituted human type antibody in which the FR portion and the constant region portion are changed to human-derived antibodies while leaving R),
As an example of the human type antibody of the L-6R antibody, PCT International Publication No. WO 92/19759 may be mentioned.

The anti-IL6R antibody of the present invention can be selected depending on the species of the animal to which it is administered, as a combination of species between the antigen IL-6R and antibody-producing cells. Generally, human cell-derived antibodies against human IL-6R are desirable for humans, and mouse cell-derived antibodies against mouse IL-6R are desirable for mice, but different species are acceptable as long as clinically acceptable. It may be a combination.

As shown in Experiment 3 of Example 2, the anti-IL-6R antibody, which is the active ingredient of the bone resorption inhibitor of the present invention, shows osteoclast in the presence of mouse IL-6 and mouse soluble IL-6R. Inhibits cell formation. In addition, the anti-IL-6R antibody, which is the active ingredient of the bone resorption inhibitor of the present invention, was prepared in
As shown in Table 1, mouse IL-6 and mouse soluble IL-6.
Suppresses bone resorption in the presence of R and human IL-1α.

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

The dose varies depending on the degree of disease state, administration method and the like, and it is necessary to select an appropriate amount appropriately. Generally, in the case of human, the indicated daily dose is 4 times within the range of about 25 to 100 micrograms. The following divided doses are available. However, the bone resorption inhibitor of the present application is not limited to these doses. In addition, it has not been reported until now that the toxicity of the IL-6R antibody has any effect.

[0023]

The present invention will be specifically described below 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. Immuno.
l. 139: 780-787, 1987) to Fast Track ^TM
Double-stranded CDNA was synthesized by random priming using kit (manufactured by Invitrogen).

Further, as a primer, mouse IL-having a recognition site for the restriction enzyme BamH1 at the 5'or 3'ends
6 genes (J. Van Snick et al., Eur. J. Immunol 18: 193,
1988) including the translation initiation codon (ATG at position 34) 2
Sequences 1-43 and stop codon (TA at position 667)
An oligomer complementary to the 658th to 683rd sequences including G) was synthesized.

These primers and Gene Am
Using the p (Takara Shuzo) kit, DNA Thermo
Cycler (Takara Shuzo) 94 ° C for 1 minute, 50 ° C
PCR was carried out for 50 minutes at 72 ° C. for 3 minutes for 2 minutes. The amplified fragment (0.66 kb) was purified by low-melting point agarose gel electrophoresis, treated with BamH1, and treated with P
It was introduced into the UC19 vector and subcloned. After excising the mouse IL6 gene with BamH1, B
The CHO cells were integrated with amH1-treated pdR and transduced. CHO cells resistant to 50 nM MTX were selected and the culture supernatant was subjected to the experiment.

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

(2) Disclosed in Saito et al.
CHO cells producing soluble mouse soluble IL-6 receptor
Cells in IMDM medium containing 10% FCS and
The supernatant was treated with RS12 antibody and Affigel 10 gel (Ba
Using an affinity column fixed to Iorad)
Purified. Obtained mouse soluble IL-6 receptor 5
Mix 0 μg with Freund's Complete Adjuvant
-Inoculated subcutaneously into the abdomen of rats (Japan Charles River)
It was After 2 weeks, add Freund's incomplete adjuvant
I was immunized. The spleen cells were sacrificed on the 45th day, and about 2 × 10 6
⁸ 1 x 10 pieces ⁷ Individual mouse myeloma cells P3U1
50% PEG 1500 (Boehringer Mannheim)
After the cells are fused by a conventional method, the cells are treated with HAT medium.
I selected the ibridomas.

After adding the hybridoma culture supernatant to an immunoplate coated with rabbit anti-rat IgG antibody (Kappel), mouse soluble IL-6R was reacted, and then rabbit anti-mouse IL-6R antibody and alkaline phosphatase-labeled sheep anti-antibody were added. ELI with rabbit IgG antibody
Hybridomas producing an antibody against the mouse soluble IL-6 receptor were screened by the SA method.
The antibody confirmed to be produced was subcloned twice to obtain a single clone (MR16-1).

The neutralizing activity of the antibody produced by this hybridoma against mouse IL-6 was MH60. BSF2 cells
(Matsuda et al., Eur. J. Immunol. 18: 951-956, 1988) was used to investigate ³ H-thymidine incorporation. MH60. 1 × 10 ⁴ BSF2 cells / 200μ
1 / well and mouse IL-6
(10 pg / ml) and MR16-1 antibody and RS12 antibody 12.3 to 1000 ng / ml were added, and the mixture was incubated at 37 ° C, 5% CO
After culturing at 44 for ₂ hours, ³ H-thymidine (IμCi /
well) was added and the uptake after 4 hours was measured (FIG. 1).

[0030]Example 1 Sham operation was performed on 8-week-old female ddy mice.
Was ovariectomized (OVX) and sacrificed 2 weeks later. F
Α-MEM medium without enol red was used to
Bone marrow was collected from the femur and centrifuged to remove bone marrow cells.
Bone marrow fluid was used. Bone resorption activity of bone marrow⁴⁵Label with Ca
It was measured by an organ culture method using a long mouse bone. You
That is, to the mother mouse⁴⁵CaCl₂ Subcutaneous administration
By ⁴⁵Fetus with 17-day gestation of Ca-labeled forearm
5% in αMEM without phenol red
CO₂ , Organ culture was performed at 37 ° C.

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

Example 2 Co-culture system of mouse osteoblasts and bone marrow cells [Takahash
i, etc .: Endocrinology 122: 1373, 1988, Takahashi, etc .: E
ndocrinology 123: 2600, 1988] was used as an index of bone resorption.

Mouse osteoblasts were prepared by the following method. That is, the skulls aseptically taken out from 1- or 2-day-old ddy mice were placed in PBS containing 0.1% collagenase (for cell dispersion, Wako Pure Chemical Industries) and 0.2% dispase (Godoshusei) at 37 ° C. It was shaken for 10 minutes in a constant temperature bath. The floating cells were collected, a new enzyme solution was further added, and the enzyme treatment was performed for 10 minutes. This enzymatic digestion 5
Repeated times, cells floating in the second to fifth digestion were collected as osteoblasts.

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

The co-cultivation was carried out by adding 1 × to a 48-well culture plate.
10 ⁴ cells / 0.5 m / well of osteoblasts and 2 × 10
Add ⁵ cells / well of bone marrow cells, 37 ° C, 5% C
They were cultured for 6-7 days at O _2. The formed osteoclasts were identified by staining with tartrate-resistant acid phosphatase (TRAP), which is a marker enzyme for osteoclasts [Ta.
kahashi, et al .: Endocrinology 122: 1373, 1988].

Mouse IL-6 or mouse soluble IL-6R [IL-6sR (sR324)] was added to these culture systems.
[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) were added to perform the following experiment. It was In addition, mouse IL-6 and sR32
For No. 4, the culture supernatants of cells obtained by incorporating these genes into CHO cells were used. Mouse IL- in culture supernatant
The respective concentrations of 6 and IL-6sR324 were measured by an enzyme immunoassay, diluted with α-MEM to an appropriate concentration, and used in the experiment.

[0037]Experiment 1 Mouse IL-6 or mouse soluble
Osteoclast formation experiments with IL-6R alone Mouse IL-6 (0.2 ng / ml-200 ng / ml) and sR
Osteoclast formation with 324 (0.5-500 ng / ml) alone
The ability to form osteoclasts was shown by itself.
There wasn't. In addition, in this experimental system
Sexual vitamin D ₃ Showed significant osteoclast formation (Fig.
3).

Experiment 2 Mouse IL-6 and mouse soluble I
Osteoclast formation experiment in the coexistence of L-6R was carried out with 20 ng / ml or 200 ng / ml of mouse IL-6.
When the osteoclast-forming ability when coexisting with 05 ng / ml to 500 ng / ml of sR324 was examined, significant osteoclast formation was observed depending on the concentration of sR324 (FIG. 4).
When the formed osteoclasts were cultured on ivory slices, many resorption cavities were observed, which were suppressed by the addition of calcitonin. Therefore, the osteoclasts formed in this experimental system were considered to be mature osteoclasts having bone resorption ability and calcitonin receptor.

Experiment 3 Mouse IL-6 and mouse soluble I
Mau for osteoclast formation in the presence of L-6 receptor
Anti-IL-6 receptor antibody (MR16-1 or R
S12) or the inhibitory effect of mouse anti-IL-6 antibody 6 to 20 ng / ml or 200 ng / ml of mouse IL-6
The osteoclast-forming ability when coexisting with 2.5 ng / ml to 500 ng / ml of sR324 was examined.
Remarkable osteoclast formation was observed depending on the concentration of R324. MR16-1 showed a concentration-dependent inhibitory effect on osteoclast formation by 20 ng / ml mouse IL-6 and 500 ng / ml sR324 in the concentration range of 1 ng / ml to 100 ng / ml. RS12 and mouse anti-IL-6 antibody showed inhibitory effects at 10 ng / ml and 100 ng / ml (Fig. 5).

Example 3 Mouse IL-6, mouse soluble
Induction in the presence of IL-6 receptor and human IL-1α
Anti-mouse IL-6 receptor anti-induced bone resorption
Inhibitory effect of the body (MR16-1) by subcutaneous administration of ⁴⁵ CaCl ₂ to mother mice
A calvaria labeled with ⁴⁵ Ca was collected from a fetus aged 16 days of gestation, and 5% C was measured in BGJb medium containing 1 mg / ml BSA.
Organ culture was performed at O ₂ and 37 ° C. After 24 hours, the medium was replaced with a new medium, and 100 pg / ml of human IL-1α, 20 ng /
ml mouse IL-6 and 500 ng / ml mouse soluble IL-6R (sR324) were added, and 37 ° C, 5% C
Cultured in O ₂ for 5 days. In this case, 10 μg / ml of the test substance MR16-1 was added to the fresh medium. Bone resorption caused by this experimental system was performed with 10 μg / ml of MR1.
6-1 suppressed almost completely (Fig. 6).

[0041]

INDUSTRIAL APPLICABILITY As described above, the bone resorption inhibitor of the present invention comprising an anti-IL-6R antibody as an active ingredient comprises IL-6 and IL-6.
It is proved that it suppresses the formation of osteoclasts in the presence of R and has an effect of inhibiting bone resorption. Therefore, the bone resorption inhibitor of the present invention, various bone metabolic diseases involved in bone resorption, such as osteoporosis, rheumatoid arthritis, multiple myeloma, neoplastic hypercalcemia, renal bone dystrophy, pazetosis, bone Expected as a therapeutic agent for metastases, osteosarcoma, etc.

[Brief description of drawings]

FIG. 1 shows MH60. FIG. 6 is a graph showing the effect of antibodies MR16-1 and RS12 on IL-6 dependent proliferation of BSF2 cells.

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 shows IL-6 or soluble I on the formation of osteoclasts in a co-culture system of mouse osteoblasts and bone marrow cells.
It is a graph which shows the effect by L-6R (sR324) alone.

FIG. 4 shows IL-6 or sR32 for the formation of osteoclasts in a co-culture system of mouse osteoblasts and bone marrow cells.
It is a graph which shows the effect of combination use of No. 4.

FIG. 5 shows IL-6 and IL-6R (sR32
4 is a graph showing the inhibitory effect of IL-6R antibody on the formation of osteoclasts by co-culturing mouse osteoblasts and bone marrow cells in the coexistence of 4).

FIG. 6 shows IL-6, soluble IL-6R (sR3.
24) is a graph showing the inhibitory effect of anti-IL6R antibody on ⁴⁵ Ca release in the coexistence of 24) and IL-1α.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Chisato Miyaura 4-24-32 Migamioka, Midori-ku, Yokohama, Kanagawa Prefecture (72) Inventor Tatsuo Suda 1-8-5, Wakaba-cho, Tachikawa-shi, Tokyo

Claims (3)

[Claims] 1. A bone resorption inhibitor containing an antibody against the interleukin 6 receptor as an active ingredient. 2. The antibody against the interleukin 6 receptor is derived from mouse.
The bone resorption inhibitor as described. 3. The bone resorption inhibitor according to claim 1 or 2, wherein the interleukin 6 receptor is derived from a mouse.