KR20070035482A - Therapeutic agent for auris interna disorder containing il-6 antagonist as active ingredient - Google Patents

Abstract

Agents for treating and / or preventing inner ear disorders comprising IL-6 antagonists, preferably anti-IL-6R antibodies as active ingredients.

Inner ear disorders, IL-6 antagonists, sensorineural hearing loss

Description

Inner ear disorder treatment containing interlockin-6 antagonist as active ingredient {THERAPEUTIC AGENT FOR AURIS INTERNA DISORDER CONTAINING IL-6 ANTAGONIST AS ACTIVE INGREDIENT}

The present invention relates to a therapeutic and / or prophylactic agent for inner ear disorder. The therapeutic and / or prophylactic agent of the present invention comprises an interleukin-6 (IL-6) antagonist.

Hearing loss caused by maze (snail inertia) or aftersight (8th cranial nerve) is called sensorineural hearing loss. There are various causes of sensorineural hearing loss, for example, Meniere's disease, drug inner ear disorders (inner ear disorders caused by antiglycologic agents such as aminoglycosides and cisplatin), viral otitis media , Purulent otitis media, temporal bone fractures, auditory nerve tumors. Incidental hearing loss, senile hearing loss, and noise hearing loss are also included in sensorineural hearing loss. Noise-induced hearing loss results in damage to the inner ear by loud noises from chain saws, internal combustion engines, heavy machinery, guns, and airplanes, and is also associated with fire, snowmobiles, air travel, rock concerts, and the like.

There are many possible causes of sensorineural hearing loss, but it is known that the acute immune response of the inner ear causes permanent hearing loss (Satoh H et al., Laryngoscope. 2002, Sep; 112 ( 9): 1627-34). In the immune system in the cochlea resulting from the injection of keyhole limpet hemocyanin (KLH) into the inner ear or subarachnoid membrane, TNF-α and IL-1β are expressed so that TNF-α exacerbates cochlear disease and a TNF-α inhibitor It has been reported that the deterioration of hearing can be partially suppressed by the above (the same document). However, there has been no report on the contribution of IL-6 to sensorineural hearing loss.

Non-patent document 1; Satoh H. et al., Laryngoscope. 2002 Sep; 112 (9): 1627-34.

It is an object of the present invention to provide a novel pharmaceutical composition for the treatment and / or prevention of endopathy.

As a result of intensive studies, the present inventors have found that IL-6 is involved in the pathogenesis of sensorineural hearing loss, and that IL-6 antagonists have a therapeutic effect on sensorineural hearing loss.

Accordingly, the present invention provides an agent for treating and / or preventing inner ear disorders containing IL-6 antagonist as an active ingredient.

The inner ear disorder is, for example, sensorineural hearing loss, wherein the sensorineural hearing loss is sensory nerve hearing loss caused by, for example, Meniere's disease, drug inner ear disorder, viral otitis, purulent otitis, temporal bone fracture or auditory nerve tumor. Or hearing loss, senile hearing loss or noise-induced hearing loss.

Optionally, the inner ear disorder is, for example, a vestibular disorder, which is due to, for example, Meniere's disease, vestibular neuritis or pharmacologic disorder.

The IL-6 antagonist is, for example, an antibody against the IL-6 receptor. This IL-6 receptor antibody is for example a monoclonal antibody against the IL-6 receptor, preferably a monoclonal antibody against the human IL-6 receptor, or a monoclonal antibody against the mouse IL-6 receptor. Can be. The monoclonal antibody to the human IL-6 receptor is, for example, a PM-1 antibody, and the monoclonal antibody to the mouse IL-6 receptor is, for example, an MR16-1 antibody.

The antibody against the IL-6 receptor is preferably a recombinant antibody. This recombinant antibody is, for example, a chimeric antibody, humanized antibody or human antibody. This humanized antibody is, for example, a humanized PM-1 antibody.

The present invention can also be described as follows.

  [1] Use of IL-6 antagonists for the manufacture of therapeutic and / or prophylactic agents for inner ear disorders.

  [2] The use according to [1], wherein the inner ear disorder is sensorineural hearing loss.

  [3] The use according to [2], wherein the sensorineural hearing loss is sensory nerve hearing loss caused by Ménière's disease, drug internal ear disorder, viral otitis media, purulent otitis media, temporal bone fracture or auditory nerve tumor.

  [4] The use according to [2], wherein the sensorineural hearing loss is sudden hearing loss, senile hearing loss, or noise deafness.

  [5] The use according to [1], wherein the inner ear disorder is vestibular disorder.

  [6] The use according to [5], wherein the vestibular disorder is vestibular disorder caused by Ménière's disease, vestibular neuritis, or pharmacological inner ear disorder.

  [7] The use according to any one of [1] to [6], wherein the IL-6 antagonist is an antibody against the IL-6 receptor.

  [8] The use according to [7], wherein the antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor.

  [9] The use according to [8], wherein the antibody against the IL-6 receptor is a monoclonal antibody against the human IL-6 receptor.

  [10] The use according to [8], wherein the antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor.

  [IL] The use according to any one of [7] to [10], wherein the antibody against the IL-6 receptor is a recombinant antibody.

  [12] The use according to [9], wherein the monoclonal antibody against the human IL-6 receptor is a PM-1 antibody.

  [13] The use according to [10], wherein the monoclonal antibody to the mouse IL-6 receptor is an MR16-1 antibody.

  [14] The use according to any one of [7] to [13], wherein the antibody to the IL-6 receptor is a chimeric antibody, a humanized antibody, or a human antibody to the IL-6 receptor.

  [15] The use according to [14], wherein the humanized antibody against the IL-6 receptor is a humanized PM-1 antibody.

The invention may also be described as follows.

  [1] A method for treating and / or preventing inner ear disorder comprising administering IL-6 antagonist.

  [2] The method of [1], wherein the inner ear disorder is sensorineural hearing loss.

  [3] The method according to [2], wherein the sensorineural hearing loss is sensory nerve hearing loss caused by Ménière's disease, drug internal ear disorder, viral otitis media, purulent otitis media, temporal bone fracture or auditory nerve tumor.

  [4] The method of [2], wherein the sensorineural hearing loss is sudden hearing loss, senile hearing loss, or noise deafness.

  [5] The method of [1], wherein the inner ear disorder is vestibular disorder.

  [6] The method according to [5], wherein the vestibular disorder is vestibular disorder caused by Ménière's disease, vestibular neuritis, or pharmacological inner ear disorder.

  [7] The method of any one of [l] to [6], wherein the IL-6 antagonist is an antibody against the IL-6 receptor.

  [8] The method of [7], wherein the antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor.

  [9] The method of [8], wherein the antibody to the IL-6 receptor is a monoclonal antibody to the human IL-6 receptor.

  [10] The method of [8], wherein the antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor.

  [11] The method of any of [7] to [10], wherein the antibody against the IL-6 receptor is a recombinant antibody.

  [12] The method of [9], wherein the monoclonal antibody against the human IL-6 receptor is a PM-1 antibody.

  [13] The method of [10], wherein the monoclonal antibody to the mouse IL-6 receptor is an MR16-1 antibody.

  [14] The method according to any one of [7] to [13], wherein the antibody to the IL-6 receptor is a chimeric antibody, humanized antibody, or human antibody against IL-6 receptor l.

  [15] The method of [14], wherein the humanized antibody against the IL-6 receptor is a humanized PM-1 antibody.

Figure 1 shows the results of Example 1, in the deafness model of mice made by three kinds of frequencies, the anti-IL-6R humanized antibody (MR16-1) administration group and mouse immunoglobulin G administration control group of the present invention It is a graph showing the decrease in hearing threshold level (dB).

FIG. 2 is an electrophoretic diagram showing the results of RT-PCR of Example 2 showing changes over time of various types of cytokines in the cochlea after an acoustic load is applied to SD rats.

Fig. 3 is a graph showing the results of quantitative RT-PCR in Example 2 showing the change over time of TNFα in the cochlea after acoustic load on SD rats.

Fig. 4 is a graph showing the results of quantitative RT-PCR in Example 2 showing the change over time of IL-6 in the cochlea after acoustic load on SD rats.

Fig. 5 is a graph showing the results of quantitative RT-PCR in Example 2 showing the change over time of IL-1β in the cochlea after acoustic loading on SD rats.

FIG. 6 is a photographic diagram showing the results of detecting IL-6 using anti-mouse IL-6 antibody using the Vectastein ABC kit in the tissues of SD rats after 6 hours of acoustic load in Example 2. FIG.

FIG. 7 is a photographic diagram showing the results of detecting IL-6 using anti-mouse IL-6 antibody using the Vectastein ABC kit in the tissues of SD rats after 6 hours of acoustic load in Example 2. FIG.

FIG. 8 shows C57BL / 6J mice loaded with 124 dB of excessive sound for 2 hours, followed by administration of MR16-1 (anti-IL-6 R humanized antibody prepared in Reference Example 4) or rat IgG (control) of the present invention. Electrophoresis showing the results of measuring the expression of total STAT3, Erk and Akt, and phosphorylated STAT3, Erk and Akt in the inner ear Corti organs.

The therapeutic agent of the present invention has an effect on suppressing the progression of tissue impairment of the cochlea, that is, hearing loss in endometrial sensorineural hearing loss. In particular, the therapeutic agent of the present invention has an effect on suppressing hearing loss in the high frequency region in sensorineural hearing loss. Like sensorineural hearing loss, vestibular organ disorders include disorders related to hair cell disorders. In sensorineural hearing loss, the cochlea is impaired, and the cochlea and vestibular organs are responsible for different senses, but the structure of hair cells and supporting cells is wrapped in lymph fluid, which is responsible for the physiological function of similar tissue construction and its physical characteristics. have. That is, based on the difference between whether the hair cell senses the negative vibration or the movement of the lymph fluid due to the acceleration, the structure senses the different senses by a similar mechanism. Disorders of the cochlea and vestibular organs are all linked to impaired function, and the sensitivity to drugs is very similar. Therefore, the therapeutic agent of the present invention is useful for the treatment of inner ear disorders such as sensorineural hearing loss and vestibular disorders.

IL-6 is a cytokine, also called B cell stimulator 2 (BSF2) or interferon β2. IL-6 has been discovered as a differentiation factor involved in the activation of B-lymphocyte cells (Hirano, T. et al., Nature (1986) 324, 73-76) and subsequently affects the function of various cells. Glucose was found to be a multifunctional cytokine (Akira, S. et al., Adv. In Immunology (1993) 54, 1-78). It has been reported that IL-6 induces maturation of T-lymphocyte cells (Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1258).

IL-6 delivers its biological activity through two proteins in the cell. One is the IL-6 receptor of a ligand-binding protein with a molecular weight of about 80 kD to which IL-6 binds (Taga, T. et al., J. Exp. Med. (1987) 166, 967-981; Yamasaki, K. et al., Science (1987) 241, 825-828). In addition to the membrane-bound type of IL-6 receptor that penetrates the cell membrane and is expressed on the cell membrane, it also exists as a soluble IL-6 receptor mainly composed of its extracellular domain.

Another is the membrane protein gp130, which has a molecular weight of about 130 kD, associated with non-ligand binding signal transduction. IL-6 and IL-6 receptors form an IL-6 / IL-6 receptor complex and then bind to gp130 to allow the biological activity of IL-6 to be transferred into cells (Taga, T. et al., Cell ( 1989) 58, 573-581).

IL-6 antagonists are substances that inhibit the transfer of biological activity of IL-6. To date, antibodies to IL-6 (anti-IL-6 antibodies), antibodies to IL-6 receptors (anti-IL-6 receptor antibodies), antibodies to gp130 (anti-gp130 antibodies), IL-6 modified variants , IL-6 or IL-6 receptor partial peptides and the like are known.

There are several reports regarding anti-IL-6 receptor antibodies (Novick, D. et a1., Hybridoma (1991) 10, 137-146, Huang, YW et al, Hybridoma (1993) 12, 621-630, International Publication No. WO95-09873, French Patent Application FR 2694767, US Patent US521628). Human type obtained by implanting mouse antibody PM-1 (Hirata, Y. et a1, J. Immunology (1989) 143, 2900-2906), one of them, the complementarity determining region (CDR) A purified PM-1 antibody is known (International Patent Application Publication No. WO92-19759).

The IL-6 antagonist is preferably an antibody against the IL-6 receptor, even more preferably a monoclonal antibody against the human IL-6 receptor or a monoclonal antibody against the mouse IL-6 receptor. to be. The monoclonal antibody to the human IL-6 receptor may be exemplified by the PM-1 antibody, and the monoclonal antibody to the mouse IL-6 receptor may be MR 16-1 antibody. Said antibody is preferably a chimeric antibody, a humanized antibody or a human antibody, for example, a humanized PM-1 antibody. Humanized antibodies are altered antibodies, also referred to as humanized antibodies or reshaped human antibodies.

IL-6 antagonists used in the present invention, as long as they are useful for the treatment and / or prevention of inner ear disorders, do not question their origin, type or shape.

IL-6 antagonists are substances that block signal transduction by IL-6 and inhibit the biological activity of IL-6. IL-6 antagonists are preferably substances having an inhibitory effect on any binding of IL-6, IL-6 receptor and gp130. IL-6 antagonists include, for example, anti-IL-6 antibodies, anti-IL-6 receptor antibodies, anti-gp130 antibodies, IL-6 modifications, soluble IL-6 receptor variants or IL-6 or IL- The partial peptide of 6 receptor and the low molecular weight substance which show the same activity as these are mentioned.

The anti-IL-6 antibody used by this invention can be obtained as a polyclonal or monoclonal antibody using a well-known means. As the anti-IL-6 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. The mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering. The antibody binds to IL-6, thereby inhibiting the binding of IL-6 to the IL-6 receptor, thereby blocking the delivery of IL-6 into biological cells.

Such antibodies include MH166 (Matsuda, T. et al., Eur. J. Immunol. (1988) 18, 951-956) and SK2 antibodies (Sato, K. et al., 21st Japan Immunological Society General Assembly) Academic records (1991) 21, 166).

Anti-IL-6 antibody production hybridomas can basically be made as follows using known techniques. That is, IL-6 is used as a sensitizing antigen, immunized according to a conventional immunization method, and the obtained immune cells are fused with known parent cells by a conventional cell fusion method, and monocloned by a conventional screening method. It can be made by screening raw antibody-producing cells.

Specifically, anti-IL-6 antibody can be obtained by the following method. For example, human IL-6 used as a sensitizing antigen for antibody acquisition is Eur. J. Biochem (1987) 168, 543-550, J. Immunol. (1988) 140, 1534-1541, or Argic. Biol. Obtained by using the IL-6 gene / amino acid sequence disclosed in Chem. (1990) 54, 2685-2688.

The gene array of IL-6 is inserted into a known expression vector system to transform an appropriate host cell, and then the desired IL-6 protein is purified in a known method in the host cell or in the culture supernatant. 6 protein can be used as sensitizing antigen. In addition, a fusion protein of IL-6 protein and other proteins can be used as a sensitizing antigen.

The anti-IL-6 receptor antibody used by this invention can be obtained as a polyclonal or monoclonal antibody using a well-known means. As the anti-IL-6 receptor antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. The antibody binds to the IL-6 receptor, thereby inhibiting the binding of IL-6 to the IL-6 receptor, thereby blocking the biological activity of IL-6 in the cell.

Such antibodies include MR16-1 antibodies (Tamura, T. et al. Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-1 antibodies (Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906), or AUK12-20 antibody, AUK64-7 antibody or AUK146-15 antibody (International Patent Application Publication No. WO 92-19759). Particularly preferred antibodies among them include PM-1 antibodies.

On the other hand, the PM-1 antibody-producing hybridoma cell line is PM-1, which is the first year of evaluation in the Center for Patented Biological Deposit of the Industrial Technology Research Institute of Japan (Independent Administrative Institution, Japan). On July 12, 1989, FERM BP-2998 became an international deposit under the Treaty of Budapest. In addition, the MR16-1 antibody-producing hybridoma cell system is a rat-mouse hybridoma MR16-1, and is a patent biological deposit center (1, 1 Chume, Tsukuba-shi, Ibaraki) 6) On March 13, 1989 (1997), FERM BP-5875 is an international deposit under the Budapest Treaty.

Anti-IL-6 receptor monoclonal antibody production hybridomas can basically be made as follows using known techniques. That is, the IL-6 receptor is used as a sensitizing antigen and immunized according to a conventional immunization method, and the obtained immune cells are fused with known parental cells by a conventional cell fusion method to produce monoclonal antibodies by conventional screening methods. Can be made by screening cells.

Specifically, to produce an anti-IL-6 receptor antibody, the following may be performed. For example, human IL-6 receptor used as a sensitizing antigen for antibody acquisition is European Patent Application Publication No. EP 325474, mouse IL-6 receptor is disclosed in Japanese Patent Application Laid-open No. 3-155795. By using the -6 receptor gene / amino acid sequence.

There are two types of IL-6 receptor proteins: IL-6 receptors expressed on cell membranes and IL-6 receptors (soluble IL-6 receptors) that deviate from the cell membranes (Yasukawa, K. et al., J. Biochem). (1990) 108, 673-676). Soluble IL-6 receptor antibodies are composed of substantially extracellular domains of IL-6 receptors bound to the cell membrane, resulting in a lack of transmembrane or transmembrane and intracellular regions. It differs from the type IL-6 receptor.

The IL-6 receptor protein may be used as long as it can be used as a sensitizing antigen for anti-IL-6 receptor antibody production used in the present invention.

Gene sequences of IL-6 receptors are inserted into a known expression vector system to transform appropriate host cells, and the desired IL-6 receptor proteins are purified in a known method in the host cells or in culture supernatant. Purified IL-6 receptor protein can be used as a sensitizing antigen. In addition, cells expressing the IL-6 receptor or fusion proteins of the IL-6 receptor protein with other proteins can be used as a sensitizing antigen.

E. coli, which contains the plasmid pIBIBSF2R containing the cDNA encoding the human IL-6 receptor, was issued on January 9, 1989, in the first year of 1989, by the Japan Institute of Industrial Technology Patent and Biotechnology Depository Center (Ibaraki Tsukuba City). In the 1st 1 center 1 of East 1 chome, HB10l-pIBIBSF2R is an international deposit under the Budapest Treaty under accession number FERM BP-2232.

The anti-gp130 antibody used by this invention can be obtained as a polyclonal or monoclonal antibody using a well-known means. As the anti-gp130 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. The antibody binds to gp130, thereby inhibiting the binding of the IL-6 / IL-6 receptor complex to gp130, thereby blocking the delivery of IL-6 into cells.

Such antibodies include AM64 antibody (Kokai 3-219894), 4B1 antibody and 2H4 antibody (US 5571513), B-S12 antibody and B-P8 antibody (Japanese Patent Laid-Open No. 8-291199). Call publications).

Anti-gp130 monoclonal antibody production hybridomas can basically be made as follows using known techniques. That is, gp130 is used as a sensitizing antigen, immunized according to a conventional immunization method, and the obtained immune cells are fused with known parental cells by a conventional cell fusion method, and monoclonal antibody-producing cells are obtained by conventional screening methods. Can be made by screening.

Specifically, monoclonal antibodies can be made as follows. For example, gp130 used as a sensitizing antigen for antibody acquisition can be obtained by using the gp130 gene sequence / amino acid sequence disclosed in European patent application EP411946.

After inserting the gene sequence of gp130 into a known expression vector system and transforming an appropriate host cell, the desired gp130 protein is purified in a known method in the host cell or culture supernatant, and the purified gp130 receptor protein is used as a sensitizing antigen. Can be used as Alternatively, a cell expressing gp130 or a fusion protein of gp130 protein with another protein may be used as a sensitizing antigen.

Since mammals immunized with sensitizing antigens are preferably selected in consideration of compatibility with the parental cells used for cell fusion, rodents such as mice, rats, hamsters, etc. are generally used. It is not.

Immunization of animals with sensitizing antigens is carried out according to known methods. For example, as a general method, it is performed by injecting sensitizing antigens intraperitoneally or subcutaneously of a mammal. Specifically, diluting and suspending the sensitizing antigen with an appropriate amount of PBS (Phosphate-Buffered Saline) or physiological saline or the like is mixed with an appropriate amount of a conventional adjuvant, for example, Freund's complete auxiliary solution, if desired. After emulsification, the mammal is preferably administered several times every 4 to 21 days. In addition, a suitable carrier can be used for immunization of sensitizing antigens.

After immunizing in this way and confirming that the desired antibody level in the serum rises, immune cells are taken out from the mammal to undergo cell fusion. Preferred immune cells to be cell fused include splenocytes in particular.

Mammalian myeloma cells as the other blast cells to be fused with the above immune cells are already known in various cell systems, such as P3X63Ag8.653 (Kearney, JF et al. J. Immunol. (1979) 123, 1548-1550). , P3X63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immuno1. (1976) 6, 511-519), MPC-11 (Margulies. DH et a1., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et a1., Nature (1978) 276, 269-270), F0 (de St. Groth, SF et al., J. Immunol.Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et a1. , Nature (1979) 277, 131-133) and the like are appropriately used.

Cell fusion of the immune cells and myeloma cells is basically carried out according to known methods such as, for example, methods such as Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). Can be run.

More specifically, the cell fusion is carried out in a conventional nutrient culture medium, for example in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used, and in order to increase the fusion efficiency, it may be used by adding an auxiliary agent such as dimethyl sulfoxide.

The use ratio of immune cells and myeloma cells is preferably 1 to 10 times the immune cells relative to the myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the proliferation of the myeloma cell system, MEM culture medium, and other conventional culture mediums used for cell culture of this kind can be used, and also fetal calf serum (FCS) and the like can be used. Serum supplements may also be used in combination.

In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution heated to about 37 ° C in advance, for example, a PEG solution having an average molecular weight of 1000 to 6000, is usually 30 to 60% (w / v) is added at the concentration and mixed to form a target fusion cell (hybridoma). Subsequently, the cell culture agent or the like which is undesirable for the growth of the hybridoma can be removed by adding the appropriate culture solution in order and repeating the centrifugation to remove the supernatant.

The hybridomas are selected by culturing in a conventional selective culture medium, for example, a HAT culture medium (culture solution containing hippoxanthine, aminopterin and thymidine). Cultivation in the HAT culture solution is generally continued for days to weeks, which is a period sufficient to kill cells (non-fusion cells) other than the target hybridoma. The conventional limiting dilution method is then performed, and screening and cloning of hybridomas producing the desired antibody is performed.

In addition to immunizing antigens with animals other than humans to obtain the above hybridomas, human lymphocytes are sensitized with desired antigen proteins or antigen expressing cells in vitro, and sensitized B lymphocytes are human myeloma cells, for example, U266 family. By fusing, a desired human antibody having binding activity to a desired antigen or antigen-expressing cell can also be obtained (see Japanese Patent Publication No. 1-59878). In addition, the desired human antibody can be obtained by administering an antigen or antigen-expressing cells to a transgenic animal having a repertoire of human antibody genes (WO 93/12227, WO 92/03918, WO 94 /). 02602, WO 94/25585, WO 96/34096, WO 96/33735)

The monoclonal antibody-producing hybridoma thus constructed can be subcultured in a conventional culture solution and can be stored long term in liquid nitrogen.

In order to obtain a monoclonal antibody from the hybridoma, a method of culturing the hybridoma in a conventional manner and obtaining it as a culture supernatant, or by administering the hybridoma to a mammal compatible with the same, multiplying the plurality The method obtained as (ascite), etc. are employ | adopted. The former method is suitable for obtaining high purity antibodies, while the latter method is suitable for mass production of antibodies.

For example, anti-IL-6 receptor antibody producing hybridomas can be constructed using the method disclosed in Japanese Patent Laid-Open No. 3-139293. To the Patent Biological Deposit Center (1st Central 1, 1, 1-chome, Tsukuba-shi, Ibaraki, Japan), on July 12, 1989, in the Treaty of Budapest as FERM BP-2998. The above-mentioned internationally deposited PM-1 antibody-producing hybridomas are injected into the abdominal cavity of BALB / c mice to obtain ascites, and the method for purifying the PM-l antibody from the ascites or the appropriate medium, for example, Cultured in 10% calf serum, RPMI1640 medium containing 5% BM-Condimed HI (manufactured by Boehringer Mannheim), hybridoma SFM medium (manufactured by GIBCO-BRL), PFHM-II medium (manufactured by GIBCO-BRL), and the culture supernatant It can be carried out by a method for purifying the PM-1 antibody from.

In the present invention, as a monoclonal antibody, a recombinant antibody produced by using a recombinant gene technique in which an antibody gene is cloned from a hybridoma, assembled into a suitable vector, and then introduced into a host can be used (for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, published in the United Kingdom by MACMIILAN PUBLISHERS LTD. 1990).

Specifically, mRNA encoding the variable (V) region of the antibody of interest is isolated from antibody-producing cells such as hybridomas. Isolation of mNA is a known method, for example, guanizin ultracentrifugal method (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. (1987) 162, 156-159) and the like, and then mRNA is prepared from all RNA using an mRNA purification kit (Pharmacia). In addition, mRNA can be directly prepared by using the QuickPrep mRNA purification kit (manufactured by Pharmacia).

From the mRNA thus obtained, cDNA of the antibody V region is synthesized using a reverse transcriptase. Synthesis of cDNA can be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit. In addition, 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and 5'-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA (1988) to perform cDNA synthesis and amplification 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). The target DNA fragment is purified from the obtained PCR product and linked to the vector DNA. Then, a recombinant vector is prepared, introduced into E. coli and the like to select colonies to produce the desired recombinant vector. The base sequence of the target DNA is confirmed by a known method such as the deoxy method.

When DNA encoding the V region of the target antibody is obtained, the DNA is ligated to the DNA encoding the desired antibody normal region (C region) and incorporated into the expression vector. Alternatively, the DNA encoding the V region of the antibody can be incorporated into an expression vector containing the DNA of the antibody C region.

In preparing the antibody used in the present invention, as described later, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, the host cell can be transformed with this expression vector to express the antibody.

In the present invention, a genetically modified genetic antibody, such as a chimeric antibody, a humanized antibody, or a human antibody, may be used for the purpose of reducing heterologous antigenicity to humans. . These modified antibodies can be produced using conventional methods.

The chimeric antibody is obtained by linking a DNA encoding the antibody V region obtained as described above to a DNA encoding a human antibody C region, integrating the same into an expression vector, and introducing the same into a host for production (European Patent Application EP 125023). , International patent application WO 92-19759). Using this conventional method, the chimeric antibody useful for this invention can be obtained.

For example, a plasmid containing DNA encoding the L and H chains of the chimeric PM-1 antibody is named pPM-k3 and pPM-h1, respectively, and the E coli having this plasmid is National Collections of Industrial. and Marine Bacteria Limited (Ferguson Building, Craibst one Estate, Bucksburn, Aberdeen AB21 9 YA, United Kingdom) on February 12, 1991, NCIMB 40366 and NCIMB 40362, respectively, in international deposits.

Humanized antibodies are implanted with complementarity determining regions (CDRs) of antibodies from mammals other than humans, such as mice, also called reconstituted human antibodies, to the complementarity determining regions of human antibodies. Recombinant DNA technology is also known (see European patent application EP 125023, International patent application WO 92-19759).

Specifically, a DNA sequence designed to connect the CDRs of a mouse antibody and a framework region (FR) of a human antibody is synthesized by PCR from several oligonucleotides having a portion overlapping with a terminal portion. The obtained DNA is ligated to the DNA encoding the human antibody C region, merged into the next expression vector, introduced into the host, and produced. (See European Patent Application EP 239400, International Patent Application WO 92-19759).

The FR of human antibody ligated through CDRs is selected to form an antigen binding site having a good complementarity determining region. If necessary, the amino acid of the framework region of the variable region of the antibody can be substituted so that the complementarity determining region of the reconstituted human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

Human antibody C regions are used in chimeric and humanized antibodies. Examples of the human antibody C region include Cγ, and for example, Cγ1, Cγ2, Cγ3 or Cγ4 can be used. In addition, the human antibody C region may be modified to improve the stability of the antibody or production of the antibody.

Chimeric antibodies consist of variable regions of mammals derived from non-human antibodies and C regions derived from human antibodies, and humanized antibodies comprise complementarity determining regions, framework regions and C regions derived from human antibodies from mammals derived from humans other than humans. It is composed of these compounds, and since the antigenicity in the human body is lowered, they are useful as antibodies for use in the present invention.

Preferred specific examples of humanized antibodies used in the present invention include humanized PM-1 antibodies (see International Patent Application Publication No. WO 92-19759).

As a method for acquiring a human antibody, in addition to the method described above, a technique for acquiring a human antibody by panning using a human antibody library is also known. For example, a phage that binds to an antigen can be selected by expressing the variable region of a human antibody on the surface of the phage by phage display as a single chain antibody (scFv). By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody bound to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is found, human antibodies can be obtained by constructing the appropriate expression vector. These methods are already well known and can be referred to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388.

The antibody gene constructed as mentioned above can be expressed and acquired by a well-known method. In the case of mammalian cells, it can be expressed by a commercially available promoter, an antibody gene to be expressed, DNA which functionally binds a poly A signal in the 3 ′ downstream thereof, or a vector comprising the same. For example, as a promoter / enhancer, a human cytomegalovirus immediate early promoter / enhancer is mentioned.

In addition, as a promoter / enhancer which can be used for antibody expression used in the present invention, viral promoter / enhancer such as retrovirus, polyoma virus, adenovirus, simian virus 40 (SV40), or human kidney factor 1 (HEF1α) Promoter / enhancer derived from such mammalian cells can be used.

For example, when using the SV40 promoter / enhancer, Mulligan et al. (Mulligan, RC et al., Nature (1979) 277, 108-114), and when using the HEF1α promoter / enhancer, Mizushima et al. It can be easily carried out according to the method (Mizushima, S. and Nagata, S., Nucleic Acids Res. (1990) 18, 5322).

In the case of E. coli, it is possible to express functionally by combining a commercially available useful promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed. For example, as a promoter, a lacZ promoter and an araB promoter can be mentioned. When using the lacZ promoter, Ward et al. Method (Ward, ES et al., Nature (1989) 341, 544-546; Ward, ES et al. FASEB J. (1992) 6, 2422-2427), using the araB promoter, by Better et al. Method (Better, M. et al. Science (1988) 240, 1041-1043).

As a signal sequence for secretion of the antibody, when produced in the outer cortex of E. coli, pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379-4383) may be used. After separating the antibody produced in the foreign material, the structure of the antibody is appropriately refolded and used (see, for example, WO96 / 30394).

As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can be used, and for the amplification of the number of gene copies in the host cell system, the expression vector is aminoglyco as a selection marker. Seed phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, and the like. .

Any production system can be used for the preparation of the antibodies used in the present invention. Production systems for antibody production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

In the case of using eukaryotic cells, there are production systems using animal cells, plant cells, or fungal cells. Examples of the animal cells include (1) mammalian cells, for example, CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, and the like. (3) For example, sf9, sf21, Tn5 and the like which are insect cells are known. As plant cells, cells derived from tobacco (Nicotiana tabacum) are known, and callus culture may be performed.

Fungal cells include yeast, for example Saccharomyces, for example Saccharomyces cerevisiae, filamentous fungus, for example, Aspergillus, for example Aspergillus. AspergiILus niger is known.

In the case of using prokaryotic cells, there is a production system using bacterial cells. Bacterial cells are known as E. coli, Bacillus subtilis.

Antibodies are obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro. Cultivation is performed by a well-known method. For example, DMEM, MEM, RPMI1640, IMDM can be used as the culture medium, and serum supplements such as calf fetal serum (FCS) can be used in combination. In addition, the antibody can be produced in vivo by introducing cells into which the antibody gene is introduced into the abdominal cavity of an animal.

On the other hand, the in vivo production system includes a production system using animals or a production system using plants. In the case of using animals, there are mammalian animals and production systems using insects.

As mammals, goats, pigs, sheep, mice, cattle and the like can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Silkworms can also be used as insects. When using plants, for example tobacco may be used.

Antibody genes are introduced into these animals or plants to produce and recover antibodies in the body of the animals or plants. For example, an antibody gene is inserted into a gene encoding a protein that is uniquely produced in milk such as goat β casein, and produced as a fusion gene. DNA fragments containing the fusion gene into which the antibody gene is inserted are injected into goat embryos, and the embryos are introduced into female goats. Desired antibodies are obtained from the milk produced by transgenic goats or their offspring born to goats that have received embryos. In order to increase the amount of milk containing the desired antibodies produced in transgenic goats, hormones can be used appropriately for transgenic goats (Ebert, KM et al., Bio / Technology (1994) 12, 699-702 ).

When silkworms are used, baculoviruses containing the desired antibody genes are infected with silkworms to obtain desired antibodies from the silkworm body fluids (Maeda, S. et al., Nature (1985) 315, 592-). 594). When tobacco is used, the desired antibody gene is inserted into a plant expression vector, for example, pMON 530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens. The bacteria are infected with tobacco, for example Nicotiana tabacum, to obtain the desired antibody from the tobacco leaves (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138). ).

As described above, when antibodies are produced in an in vivo or in vitro production system, DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) are respectively incorporated into an expression vector to simultaneously transform the host. Alternatively, the host can be transformed by incorporating DNA encoding the H and L chains into a single expression vector (see International Patent Application Publication No. WO 94-11523).

The antibody used in the present invention may be a fragment of the antibody or a modification thereof as long as it can be suitably used in the present invention. For example, as a fragment of an antibody, Fab, F (ab ') ₂ , Fv, or the single chain Fv (scFv) which ligated Fv of H chain and L chain with the appropriate linker is mentioned.

Specifically, antibodies are treated with enzymes such as papain or pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed, introduced into expression vectors, and expressed in appropriate host cells. (See, eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, AH, Methods in Enzymo1ogy (1989) 178, 476-496; Plueckthun, A. & Skerra, A., Methods in Enzymology (1989) 178, 476-496; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121 , 663-66, Bird, RE et al., TIBTECH (1991) 9, 132-137).

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

The DNA encoding the scFv is based on a DNA encoding the H chain or the V region of the H chain of the antibody, and a DNA encoding the V region of the L chain or the L chain. Amplify the DNA portion to be encoded by PCR using a pair of primers that define both ends thereof, and then pair the primers that define the DNA encoding the peptide linker portion and its ends to be connected to the H chain and L chain, respectively. It is obtained by amplifying in combination.

Once the DNA encoding the scFv is produced, the expression vector containing them and the host transformed with the expression vector can be obtained according to a conventional method, and the scFv can be obtained by the conventional method using the host. Can be.

The fragments of these antibodies can be produced and expressed by the host by obtaining and expressing the gene in the same manner as described above. "Antibody" as used in the present invention also includes fragments of these antibodies.

As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) may be used. The "antibody" as used in the present invention also includes these antibody modifications. In order to obtain such an antibody modification, it can obtain by chemically modifying the obtained antibody. These methods are already established in this field.

The antibody produced and expressed as mentioned above can be refine | purified until it isolate | separates and isolates from a host inside and outside a cell. Isolation and purification of the antibody used in the present invention can be carried out by affinity chromatography. As a column used for affinity chromatography, a protein A column and a protein G column are mentioned, for example. As a carrier used for the Protein A column, for example, Hyper D, POROS, Sepharose F.F. Etc. can be mentioned. In addition, the separation and purification methods used in conventional proteins can be used, and there is no limitation.

For example, by appropriately combining and combining chromatography, filter, ultrafiltration, salting out, dialysis, and the like other than the affinity chromatography, the antibody used in the present invention can be separated and purified. As chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, etc. are mentioned, for example. These chromatography can be applied to high performance liquid chromatography (HPLC). Alternatively, reverse phase HPLC may be used.

The concentration measurement of the antibody obtained above can be performed by measurement of absorbance or ELISA. That is, in the case of measurement of absorbance, after dilution with PBS (-) is appropriately, the absorbance at 280 nm is measured, and 1 mg / ml is calculated as 1.35 OD. In addition, in the case of ELISA, it can measure as follows. That is, 100 μl of goat anti-human IgG (TAG product) diluted to 1 μg / ml in 0.1 M hydrogen carbonate buffer (pH 9.6) was added to a 96-well plate (Nunc product), and cultured at 4 ° C. overnight. Immobilize After blocking, 100 μl of human IgG (CAPPEL product) is added as a sample or a sample containing the antibody or antibody used in the present invention, which is diluted appropriately, and incubated at room temperature for 1 hour.

After washing, 100 µl of 5000-fold diluted alkaline phosphatase-labeled anti-human IgG (BID SOURCE) was added and incubated for 1 hour at room temperature. After washing, the substrate solution was added and incubated, and then the absorbance at 405 nm was measured using MICROP LATE READER Model 3550 (manufactured by Bio-Rad) to calculate the desired antibody concentration.

The IL-6 modified substance used in the present invention is a substance that has a binding activity with the IL-6 receptor and does not transmit the biological activity of IL-6. In other words, the IL-6 modified molecule binds IL-6 to the IL-6 receptor in a competitive manner, but blocks signal transduction by IL-6 because it does not transmit the biological activity of IL-6.

The IL-6 modified substance is produced by introducing a mutation by replacing an amino acid residue of the amino acid sequence of IL-6. IL-6, which is the basis of the IL-6 modified substance, does not matter its origin, but human IL-6 is preferable in consideration of antigenicity and the like.

Specifically, the secondary structure of IL-6 is predicted using a known molecular modeling program such as WHATIF (Vriend et al., J. Mol. Graphics (1990) 8, 52-56). And the effect on the entirety of the amino acid residue to be substituted again. After determining the appropriate amino acid residue, the gene encoding the IL-6 modified product is obtained by introducing a mutation such that the human IL-6 gene is a vector containing a code base sequence as a template and the amino acid is substituted by a commonly performed PCR method. Lose. If necessary, the IL-6 modified product can be obtained by assembling into a suitable expression vector and following the method for expression, production and purification of the recombinant antibody.

Specific examples of IL-6 modifications include those described in Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, and Savino et al., EMBO J. (1994) 13, 1357-1367, WO 96-18648, WO 96-17869.

The IL-6 partial peptide or IL-6 receptor partial peptide used in the present invention is a substance having binding activity with the IL-6 receptor or IL-6, and not transmitting the biological activity of IL-6, respectively. That is, the IL-6 partial peptide or IL-6 receptor partial peptide binds to the IL-6 receptor or IL-6, and by capturing them specifically inhibits the binding of IL-6 to the IL-6 receptor. As a result, it blocks signal transduction by IL-6 because it does not transmit the biological activity of IL-6.

The IL-6 partial peptide or IL-6 receptor partial peptide consists of the amino acid sequence of some or all of the regions involved in the binding of IL-6 and IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor. Peptides. Such peptides usually consist of 10 to 80, preferably 20 to 50, even more preferably 20 to 40 amino acid residues.

The IL-6 partial peptide or IL-6 receptor partial peptide, in the amino acid sequence of IL-6 or IL-6 receptor, specifies a region involved in binding of IL-6 to IL-6 receptor, and part or all of it The amino acid sequence of can be made by a conventionally known method, for example, by genetic engineering or peptide synthesis.

Genetic engineering of the 1L-6 partial peptide or IL-6 receptor partial peptide can be accomplished by assembling a DNA sequence encoding a desired peptide into an expression vector and expressing, producing and purifying the recombinant antibody. .

In order to make an IL-6 partial peptide or IL-6 receptor partial peptide into a peptide synthesis method, the method commonly used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method, can be used.

Specifically, the method can be carried out according to the method described in the development of genus medicines, peptide synthesis 14 (supervision Yajima Jimei, Gyeongcheon Bookstore, Japan, 1991). In the solid phase synthesis method, for example, amino acids corresponding to the C terminus of the peptide to be synthesized are bonded to a support insoluble in an organic solvent, and the amino acid protecting the α-amino group and the side chain functional group with an appropriate protecting group from the C terminus to the N terminus direction. As described above, a method of extending the peptide chain by alternately repeating the condensation reaction by one amino acid and the reaction of leaving the protecting group of the α-amino group of the amino acid or peptide bound on the resin can be used. Solid phase peptide synthesis methods are roughly classified into Boc method and Fmoc method according to the kind of protecting group used.

After the target peptide is synthesized, the deprotection reaction and cleavage reaction of the peptide chain from the support are carried out. For the cleavage reaction of the peptide chain, hydrogen fluoride or trifluoromethane sulfonic acid can be conventionally used in the Boc method, and TFA can be commonly used in the Fmoc method. In the Boc method, the protective peptide resin is treated, for example, in hydrogen fluoride in the presence of anisole, followed by removal of the protecting group and recovery of the peptide from the support. Lyophilization of this yields a crude peptide. On the other hand, in the Fmoc method, for example, deprotection reaction and cleavage reaction of the peptide chain from the support can be carried out by the above-described operation in TFA.

The obtained rough peptide can be separated and purified by HPLC. In the elution, it may be carried out under optimum conditions using a water-acetonitrile solvent commonly used for protein purification. Fractions corresponding to the peaks of the profiles of the obtained chromatography are collected and lyophilized. The peptide fraction thus purified is identified by molecular weight analysis, amino acid composition analysis, or amino acid sequence analysis by mass spectrum analysis.

Specific examples of the IL-6 partial peptide and the IL-6 receptor partial peptide are disclosed in Japanese Patent Laid-Open No. 2-188600, Japanese Patent Laid-Open No. 7-324097, Japanese Patent Laid-Open No. 8-311098, and US Patent Publication US 5210075. .

The IL-6 signal transduction inhibitory activity of the IL-6 antagonists used in the present invention can be evaluated by a commonly used method. Specifically, IL-6-dependent human myeloma (S6B45, KPMM2), human Rennert T-lymphoma cell line KT3, or IL-6-dependent cell MH60.BSF2 is cultured, and IL-6 is added thereto, and IL Activity can be measured by the incorporation of ³ H-thymidine into IL-6 dependent cells in the presence of -6 antagonists.

In addition, IL-6 receptor expressing cells in culture of U266, and the addition of ¹²⁵ I labeled IL-6, and by at the same time the addition of IL-6 antagonists, IL-6 measures the ¹²⁵ I labeled IL-6 bound to the receptor expressing cells do. In the assay system, in addition to the group in which the IL-6 receptor antagonist is present, a negative control containing no IL-6 antagonist is made, and the results obtained in both are compared to evaluate the IL-6 inhibitory activity of the IL-6 antagonist.

As shown in the following examples, the anti-IL-6 receptor antibody has been recognized as a therapeutic effect of inner ear disorder, suggesting that IL-6 antagonists such as anti-IL-6 receptor antibody are useful as therapeutic agents for inner ear disorder. .

The subject of treatment in the present invention is a mammal. The mammal to be treated is preferably a human.

The inner ear disorder treatment / prevention agent of the present invention can be administered orally or parenterally or systemically. For example, intravenous injections such as drips, intramuscular injections, intrathoracic injections, intraperitoneal injections, subcutaneous injections, suppositories, intestinal washes, oral enteric preparations, and the like may be selected. Appropriate administration method can be selected. The effective dose is selected in the range of 0.0lmg to 100 mg per kg of body weight per dose. Alternatively, a dosage of 1-100 mg, preferably 5-50 mg per patient can be selected.

Preferred dosages and administration methods, for example, in the case of anti-IL-6 receptor antibodies, are effective doses such that free antibodies are present in the blood. 0.5 mg to 40 mg, preferably lmg to 20 mg, divided into 1 to several times (for 4 weeks), for example, 2 times / week, 1 time / week, 1 time / 2 weeks, 1 time / 4 weeks, etc. It is a method of administering by the method of intravenous injection, such as drip, subcutaneous injection, etc. as an administration schedule. The dosing schedule increases the dosing interval from twice / week or once / week to twice / week, once / three weeks, once / four weeks while observing the observation of the condition and the trend of blood test value, etc. You can also adjust with.

The agent for treating and / or preventing the inner ear disorder of the present invention may contain a pharmaceutically acceptable carrier or additive according to the administration route. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble dextran Sodium carboxy starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA) ), Mannitol, sorbitol, lactose, pharmaceutically acceptable surfactant, and the like. Additives to be used may be selected from the above depending on the formulation, or may be selected in combination, but is not limited thereto.

(Example)

Hereinafter, the present invention will be described in detail with examples and reference examples, but the present invention is not limited thereto.

Example  One.

Experiment method

(1) Hearing measurement of mouse before acoustic load

Four-week-old C57BL / 6J male mice were subjected to hearing measurement by auditory brainstem response (ABR) on the day before the acoustic load. Prior to the measurement, intraperitoneal injection of Xyladine and Ketamine was used for anesthesia of sufficient depth, and additional anesthesia by Ketamine was performed as needed during the measurement.

Auditory Brainstem Response To determine the degree of noise-induced hearing loss under the noise conditions, threshold shifts of the auditory brainstem response (ABR) were tested. ABR measurements are performed using waveform storage and stimulus control in one Scope software from the PowerLab system (Model: PowerLob2 / 20, AD Instruments Castle Hill, Australia). This was done using a Digital Biomap system (model: BAL-1, Tucker-Davis Technologies FL / USA). Acoustic stimulation was generated by a coupler-type speaker (model: ESlspc, BioResearch Center Nagoya / Japan) inserted into the ear canal of the mouse. Rupture Stimulation Generates 0.1 ms rise / fall time (cosinegate) and 1 ms flat segment, and amplitudes are sound generator and attenuation real-time processor and programmable attenuator (models: RP2.1 and PA5, Tucker-Davis Technologies FL / USA). The sound level was corrected using a Sound Level Meter (Model: LA-5111, Ono Sokki Yokohama / Japan). Stainless needle electrodes were placed on the top and cavity sides of the left and right ears for recording. In general, the ABR waveform was recorded for 12.8 ms at a sampling rate of 40,000 Hz using a 50-5000 Hz bandpass filter setting and averaged from 256 stimuli at a frequency of 9 Hz. The ABR waveform was recorded at 5-dB SPL intervals down from the peak amplitude until the waveform was invisible. The hearing frequencies given for threshold measurement were measured as three types of 4 kHz, 12 kHz, and 20 kHz, and each hearing threshold was measured.

(2) generation of deafness model

In an acoustic load device (a device using masking noise of RION Audiometry AA67N as an acoustic generator, amplified by SONY SRP-P150 and FOSTEX D-1405 as an amplifier, and then loaded into a sealed space with a speaker having a diameter of about 12 cm) The mouse was put into the cage of 3 cm in height made of the metal mesh which stood still below 1 cm. The cages were radially divided by a similar network of gold, and four mice were placed in separate rooms at the same time. The loud sound was loaded for 2 hours under the condition that the sound load device was set in advance so as to take a sound pressure of 124 ± 1db (measured several times during the 2 hours load by CASELA Co., Ltd.). The sound used octave band noise of 4kHz SPL. In addition, a thermometer was placed in the acoustic device to measure the temperature before and after the acoustic load.

(3) Drug administration

After the preparation of (2), the animals were divided into two groups A and B, and 2 mg / body of rat IgG 2 mg / body or MR16-1 (anti-IL-6R humanized antibody prepared in Reference Example 4) were each quickly. It was administered to the group of. About administration, it was set as a blind test and the investigator was not informed of the administration content in each group until the hearing measurement of (4) was completed.

(4) hearing threshold measurement

In the same manner as in the above (1), hearing measurement was performed one week after the acoustic load and drug administration. After confirming sufficient anesthesia depth after the measurement, the mouse was cut, the temporal bone was extracted and fixed, and preserved for histological examination.

(5) interpretation of results

The hearing difference obtained in the above (4) of each individual and the hearing difference before the treatment obtained in the above (1) were taken to calculate the hearing threshold lowering degree (dB). The average hearing threshold drop (dB) was calculated for each frequency in each of the MR 16-1 and control groups.

result

When anesthesia was performed in (4), one animal died and was excluded because hearing measurement became impossible. The number of animals for which the hearing threshold was obtained was n = 4 in the MR16-1-administered group and n = 2 in the IgG-administered group. The temperature in the acoustic load device was 25 ° C. before the load and 27 ° C. after the load. The results are shown in Table 1 below and FIG. 1.

Processing frequency IgG administration control group MR 16-1 administration group Difference in threshold change 4kHz 12kHz 20kHz 22.5 35 55 37.5 27.5 28.8 -15 +7.5 +26.3

Review

Acoustic trauma is a physical external force called sound pressure, and the model used in this experiment can be considered as a physical tissue-damage model of the cochlea. In this experiment, excessive sound centered around a frequency of 4 kHz was loaded. Sensors that perceive sound in the cochlea are spatially tonic in frequency (tonotropic), and deafness in the control group is significant at 4 kHz, and it becomes excessive as it falls off into the high-frequency region, which is excessive in the region of 4 kHz. Because of the negative loading, the closer to this area, the greater the tissue damage.

On the other hand, in the IL-6 receptor antibody-administered group, hearing loss was suppressed as compared with the control group as 12 kHz, 20 kHz, and excessive sound were loaded and dropped from the site directly subjected to tissue damage. As mentioned above, IL-6 receptor antibody has an effect on the suppression of spatial progression of the cochlear tissue failure, ie, spatial progression of hearing loss in endometrial sensorineural hearing loss, or on the suppression of the hearing loss in the high frequency region of sensorineural hearing loss. It can be considered to have.

Example  2. Acoustic trauma  Expression of Inflammatory Cytokines in the Posterior Cochlea

Hearing loss models were made in the same manner as in Example 1 using 3 to 4 week old male SD rats. Immediately after the acoustic load, 3 hours later, 6 hours later, 12 hours later, 24 hours later, 3 days later, 5 days later, 7 days later, and 28 days later, temporal bones were removed from each rat. While keeping the lymphatic fluid in the cochlea free from leaking, only the cochlea was extracted and the expression of various inflammatory cytokines was measured by RT-PCR method. The results are shown in FIG. As can be seen from FIG. 2, the expression of TNF-α, IL-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), and IL-6 was elevated in the cochlea of the acoustic trauma model. On the other hand, expression of IL-12 p40 and GM-CSF was not recognized.

Next, the quantitative RT-PCR method measured the change over time of TNF-α, IL-6, IL-1β expression. For quantitative RT-PCR, 18S rRNA was used as a reference gene and the results were analyzed by the ΔΔCt method. The results are shown in FIGS. 3 to 5. As can be seen from FIGS. 3 to 5, expression of TNF, IL-1β, and IL-6, which showed peaks in expression levels less than 24 hours after acoustic trauma, were recognized.

In addition, frozen sections were immunohistostained using tissues for 6 hours after acoustic load. Animals used were SD rats, deafness model was prepared in the same manner as in Example 1, and the head was cut 6 hours after the noise load. The temporal bone was quickly removed, fixed for 6 hours in 4% paraformaldehyde, and demineralized with 0.5 mol / l EDTA solution to rapidly freeze the sample using liquid nitrogen. Product) to obtain a frozen section by cutting to 7㎛ thickness. This section was stained immunohistochemically. For staining, the Vectastein ABC kit was used and the color was developed using DAB solution. As staining control, staining performed without adding a primary antibody was employed. The results are shown in FIGS. 6 to 7. As can be seen in Figures 6 to 7, in the cochlea 6 hours after the acoustic load, the expression of IL-6 was found in the outer wall of the cochlea and the basement membrane just below the corti organ.

Example  3. IL -6 Antagonist  Confirmation of efficacy by systemic administration

In the same manner as in Example 1, male C57BL / 6J mice were loaded with 124 dB of excessive sound for 2 hours, and immediately following administration of MR16-1 2 mg / body or rat IgG 2 mg / body intraperitoneally. 8 hours after administration of the antibody, the temporal bone was removed, and both inner ear were extracted, and the corti organs were analyzed, and each was recovered from the left and right sides by dividing into the first apex and the second basal. Western blot was used to measure the expression of total STAT3, Erk and Akt, and phosphorylated STAT3, Erk and Akt. The results are shown in FIG. As can be seen in Figure 8, the signal of the phosphorylated STAT3, phosphorylated Erk, phosphorylated Akt strongly inhibited in the control group was suppressed in the MR16-1 administration group. From this result, it was confirmed that systemically administered MR16-1 expresses the effect in the cochlea.

According to this result and the result of Example 2, MR16-1 of the IL-6 signal expressed in the cochlea early after acoustic trauma to the mechanism of action of suppressing hearing loss by the MR16-1 intraperitoneal administration shown in Example 1 It seems that the inhibitory effect by

Reference Example  One. Preparation of Human Soluble IL-6 Receptor

Soluble IL- by PCR using the plasmid pBSF2R.236 containing the cDNA encoding the IL-6 receptor obtained according to the method of Yamasaki et al. (Yamasaki, K. et al., Science (1988) 241, 825-828). 6 receptors were prepared. Plasmid pBSF2R.236 was digested with restriction enzyme Sph I to obtain IL-6 receptor cDNA, which was inserted into mp18 (Amersham). Using synthetic oligoprimers designed to introduce stop codons into the IL-6 receptor cDNA, mutations were introduced into the IL-6 receptor cDNA by PCR using an in vitro Mutagenesis system (Amersham). This operation introduced the stop codon at the position of amino acid 345 to obtain a cDNA encoding the soluble IL-6 receptor.

To express soluble IL-6 receptor cDNA in CRO cells, it was linked with plasmid pSV (manufactured by Pharmaeia) to obtain plasmid pSVL344. The soluble IL-6 receptor cDNA cut | disconnected with Hind III-Sal I was inserted into the plasmid pECEdhfr containing the dhfr cDNA, and CHO cell expression plasmid pECEdhfr344 was obtained.

10 μg of plasmid pECEdhfr344 was converted into calcium phosphate sedimentation method (Chen, C. et al., Dhfr-CRO cell line DXB-11 (Urlaub, G. et al., Proc Natl. al., Mol. Cell. Biol. (1987) 7, 2745-2751). Transfected CHO cells were cultured for 3 weeks in nucleotide-free αMEM selective cultures containing 1 mM glutamine, 10% dialysis FCS, 100 U / ml penicillin and 100 μg / ml streptomycin.

Selected CHO cells were screened by limiting dilution to obtain a single CHO cell clone. This CHO cell clone was amplified in methotrexate at a concentration of 20 nM to 200 nM to obtain CHO cell line 5E27 producing human soluble IL-6 receptor. CRO cell line 5E27 was incubated in iscove-modified Dulbecco culture (IMDM, Gibco) containing 5% FBS. The culture supernatant was recovered and the concentration of soluble IL-6 receptor in the culture supernatant was measured by ELISA. As a result, it was confirmed that soluble IL-6 receptor is present in the culture supernatant.

Reference Example  2. Anti-human IL Preparation of --6 Antibody

Immunizing BALB / c mice with 10 μg of recombinant IL-6 (Hirano, T. et al., Immunol. Lett. (1988) 17, 41) with Freund's complete adjuvant, This was repeated every week until -IL-6 antibody was detected. Immune cells were extracted from local lymph nodes and fused with myeloma cell line P3U1 using polyethylene glycol 1500. Hybridomas were selected according to the method of Oi et al. (Selective Methods in Cellular Immunology, WHFreeman and Co., San Francisco, 351, 1980) using HAT cultures to produce anti-human IL-6 antibodies. Bridoma was established.

Hybridomas producing anti-human IL-6 antibodies were subjected to IL-6 binding assays as follows. That is, a flexible polyvinyl 96-well microplate (manufactured by Dynatech Laboratories, Inc., Alexandria, VA) was placed in 100 M of chlorine anti-mouse Ig (10 µl / g) in 0.1 M carbonate-hydrocarbonate buffer (pH 9.6). ml, Cooper Biomedical, Inc., Malvern, PA) at 4 ° C. overnight. Plates were then treated with PBS containing 100 μl of 1% bovine serum albumin (BSA) for 2 hours at room temperature.

After washing with PBS, 100 μl of hybridoma culture supernatant was added to each well and incubated at 4 ° C. overnight. Plates were washed, ¹²⁵ I labeled recombinant IL-6 was added to each well to 2000 cpm / 0.5 ng / well, and after washing the radioactivity of each well was gamma counter (Beckman Gamma 9000, Beckman Instruments, Fullerton, CA). Was measured. Of the 216 hybridoma clones, 32 hybridoma clones were positive in the IL-6 binding assay. Of these clones, finally stable MH166.BSF2 was obtained. The anti-IL-6 antibody MH166 produced by the hybridoma has a subtype of IgG1κ.

Subsequently, the neutralizing activity of the proliferation of hybridoma by MH166 antibody was investigated using IL-6 dependent mouse hybridoma clone MH60.BSF2. MH60.BSF2 cells to 1 x 10 ⁴ / 200㎕ / well so that the distribution and the addition of sample containing MH166 antibody herein is 48 hours and, 0.5μCi / ³ H- tea secluded (New England Nuclear of the well, Boston, MA) was added and the culture was continued for another 6 hours. Cells were placed on glass filter paper and treated with an automatic harvester (Labo Mash Science Co., Tokyo, Japan). Rabbit anti-IL-6 antibody was used as a control.

As a result, MH166 antibody dose-dependently inhibited the incorporation of ³ H-thymidine in MH60.BSF2 cells induced by IL-6. This made it clear that the MH166 antibody neutralizes the activity of IL-6.

Reference Example  3. Preparation of Anti-Human IL-6 Receptor Antibodies

Sepharose 4B (Pharmacia Fine Chemicals) activating the anti-IL-6 receptor antibody MT18 with CNBr prepared by the method of Hirata et al. (Hirata, Y. et al. J. Immunol. (1989) 143, 2900-2906). Product, Piscataway, NJ) and binding according to the attached regimen, purifying the IL-6 receptor (Yamasaki, K. et al., Science (1988) 241, 825-828). Human myeloma cell line U266 was treated with 1 mM p-para-aminophenyl methanesulfonyl fluoride hydrochloride (made by Wako Chemicals) containing 1% digitonin (from Wako Chemicals), 10 mM triethanolamine (pH 7.8) and 0.15 M NaCl. Toxin buffer) and mixed with MT18 antibody bound to Sepharose 4B beads. The beads were then washed six times in digittonin buffer to prepare partially purified IL-6 receptor for use in immunization.

BALB / c mouse 3 x l0 ⁹ The partially purified IL-6 receptor obtained from dog U266 cells was immunized four times every 10 days, and then hybridomas were prepared by conventional methods. Hybridoma culture supernatants from growth-positive wells were tested for binding activity to the IL-6 receptor by the following method. 5 x 10 ⁷ U266 cells were labeled with ³⁵ S-methionine (2.5 mCi) and solubilized with the digittonin buffer. Solubilized U266 cells were mixed with MT18 antibody bound with 0.04 ml volume of Sepharose 4 B beads, then washed six times in digittonin buffer, and ³⁵ S-methionine in 0.25 ml digittonin buffer (pH 3.4). The labeled IL-6 receptor was eluted and neutralized with 0.025 ml of 1M Tris (pH 7.4).

0.05 ml of hybridoma culture supernatant was mixed with 0.01 ml of Protein G Sepharose (Phramacia). After washing, Sepharose was incubated with ³⁵ S labeled IL-6 receptor solution prepared above 0.005ml. Solution precipitates were analyzed by SDS-PAGE to examine the hybridoma culture supernatants that react with the IL-6 receptor. As a result, a reactive positive hybridoma clone PM-1 (FERM BP-2998) was established. Antibodies produced from hybridoma PM-1 have a subtype of IgG1.

The inhibitory activity of IL-6 binding to human IL-6 receptor of the antibody produced by hybridoma PM-1 was investigated using human myeloma cell line U266. Human recombinant IL-6 was prepared from E. coli (Hirano, T. et al., Immunol. Lett. (1988) 17, 41-45) and ^{125 with} Bolton-Hunter reagent (New England Nuclear, Boston, Mass.). Labeled with I (Taga, T. et al., J. Exp. Med. (1987) 166, 967-981).

4 × 10 ⁵ U266 cells were incubated with culture supernatant of 70% (v / v) hybridoma PM-1 and 14,000 cpm of ¹²⁵ I labeled IL-6 for 1 hour. 70 μl of the sample was layered on 300 μl of FCS in 400 μl of microfuge polyethylene tube, centrifuged, and the radioactivity of the cells was measured.

As a result, it was found that the antibody produced by hybridoma PM-1 inhibits the binding of IL-6 to the IL-6 receptor.

Reference Example  4. Preparation of Anti-Mouse IL-6 Receptor Antibodies

Monoclonal antibodies against the mouse IL-6 receptor were prepared by the methods described in Saito, et al., J. Immunol. (1991) 147, 168-173.

CHO cells producing mouse soluble IL-6 receptors were cultured in IMDM culture containing 10% FCS, and the anti-mouse IL-6 receptor antibody RS12 (see Saito, et al., Supra) was prepared from the culture supernatant using an Affigel 10 gel ( Mouse soluble IL-6 receptor was purified using an affinity column immobilized on Biorad.

50 μg of the obtained mouse soluble IL-6 receptor was mixed with Freund's complete adjuvant and injected into the abdomen of the wister rat. Two weeks later, they were further immunized with Freund's incomplete supplement. Rat splenocytes were harvested on day 45, and approximately 2 × 10 ⁸ cells were fused in a conventional manner with 1 × 10 ⁷ mouse myeloma cells P3U1 and 50% PEG1500 (from Boehringer Mannheim) and then in HAT medium. Hybridomas were screened.

Hybridoma culture supernatants were added to plates coated with rabbit anti-rat IgG antibody (Cappel), followed by reaction with mouse soluble IL-6 receptor. The hybridomas that produce antibodies to the mouse soluble IL-6 receptor were then screened by ELISA method with rabbit anti-mouse IL-6 receptor antibody and alkaline phosphatase-labeled sheep anti-rabbit IgG. Hybridoma clones whose antibody production was confirmed were subjected to two screenings to obtain a single hybridoma clone. This clone was named MR16-1.

Neutralizing activity of the antibody produced by the hybridoma in mouse IL-6 transmission was determined using MH60.BSF2 cells (Matsuda, T. et al., J. Immunol. (1988) 18, 951-956). ³ H-thymidine incorporation was investigated. In a 96-well plate, MH60.BSF2 cells were prepared so that the 1 x 10 ⁴ gae / 200㎕ / well. 10 pg / ml of mouse IL-6 and MR16-1 antibody or RS12 antibody were added to the plate, incubated for 44 hours at 37 ° C and 5% C0 ₂ with 12.3 to 1000 ng / ml, followed by 1 μCi / well of ³ H-thymi. Dean was added. After 4 hours the incorporation of ³ H-thymidine was measured. As a result, the MR16-1 antibody was MH60. ³ H-thymidine incorporation by BSF2 cells was inhibited.

Thus, it was found that antibodies produced by hybridoma MR16-1 (FERM BP-5875) inhibit the binding of IL-6 to the IL-6 receptor.

Claims (45)

An agent for treating and / or preventing inner ear disorder comprising IL-6 antagonist as an active ingredient. The therapeutic and / or prophylactic agent according to claim 1, wherein the inner ear disorder is sensorineural hearing loss. 3. The therapeutic and / or preventive agent according to claim 2, wherein the sensorineural hearing loss is sensorineural hearing loss caused by Ménière's disease, drug internal ear disorder, viral otitis, purulent otitis, temporal bone fracture or auditory nerve tumor. 3. The therapeutic and / or prophylactic agent according to claim 2, wherein the sensorineural hearing loss is sudden hearing loss, senile hearing loss or noise deafness. The therapeutic and / or prophylactic agent according to claim 1, wherein the inner ear disorder is vestibular disorder. 6. The therapeutic and / or prophylactic agent according to claim 5, wherein the vestibular disorder is vestibular disorder caused by Ménière's disease, vestibular neuritis or drug internal ear disorder. The therapeutic and / or prophylactic agent according to any one of claims 1 to 6, wherein the IL-6 antagonist is an antibody against an IL-6 receptor. 8. The therapeutic and / or prophylactic agent according to claim 7, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor. The therapeutic and / or prophylactic agent according to claim 8, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the human IL-6 receptor. 9. The therapeutic and / or prophylactic agent according to claim 8, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor. The therapeutic and / or prophylactic agent according to any one of claims 7 to 10, wherein the antibody against the IL-6 receptor is a recombinant antibody. 10. The therapeutic and / or prophylactic agent according to claim 9, wherein the monoclonal antibody against human IL-6 receptor is a PM-1 antibody. The therapeutic and / or prophylactic agent according to claim 10, wherein the monoclonal antibody against the mouse IL-6 receptor is an MR16-1 antibody. The therapeutic and / or prophylactic agent according to any one of claims 7 to 13, wherein the antibody against the IL-6 receptor is a chimeric antibody, humanized antibody or human antibody against the IL-6 receptor. The therapeutic and / or prophylactic agent according to claim 14, wherein the humanized antibody against the IL-6 receptor is a humanized PM-1 antibody. Use of IL-6 antagonists for the manufacture of a therapeutic and / or prophylactic agent for internal disorders. Use according to claim 16, characterized in that the inner ear disorder is sensorineural hearing loss. 18. The use according to claim 17, wherein the sensorineural hearing loss is sensorineural hearing loss caused by Ménière's disease, drug inner ear disorder, viral otitis media, purulent otitis media, temporal bone fracture or auditory nerve tumor. 18. The use according to claim 17, wherein the sensorineural hearing loss is sudden hearing loss, senile hearing loss or noise deafness. Use according to claim 16, characterized in that the inner ear disorder is a vestibular organ disorder. 21. The use according to claim 20, wherein the vestibular organ disorder is vestibular organ disorder caused by Ménière's disease, vestibular neuritis or drug internal ear disorder. 22. The use of any one of claims 16 to 21, wherein the IL-6 antagonist is an antibody against an IL-6 receptor. The use of claim 22, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor. The use according to claim 23, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the human IL-6 receptor. The use according to claim 23, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor. The use according to any one of claims 22 to 25, wherein the antibody against the IL-6 receptor is a recombinant antibody. The use of claim 24, wherein the monoclonal antibody against the human IL-6 receptor is a PM-1 antibody. The use of claim 25, wherein the monoclonal antibody against the mouse IL-6 receptor is an MR 16-1 antibody. The use according to any one of claims 22 to 28, wherein the antibody against the IL-6 receptor is a chimeric antibody, humanized antibody or human antibody against the IL-6 receptor. The use of claim 29, wherein the humanized antibody against the IL-6 receptor is a humanized PM-1 antibody. A method for treating and / or preventing endopathy comprising administering an IL-6 antagonist to a subject. 32. The method of claim 31 wherein the inner ear disorder is sensorineural hearing loss. 33. The method of claim 32, wherein the sensorineural hearing loss is sensorineural hearing loss caused by Ménière's disease, drug internal ear disorder, viral otitis, purulent otitis, temporal bone fracture or auditory nerve tumor. 33. The method of claim 32, wherein the sensorineural hearing loss is sudden hearing loss, senile hearing loss or noise deafness. 32. The method of claim 31 wherein the inner ear disorder is vestibular disorder. 36. The method of claim 35, wherein the vestibular disorder is vestibular disorder caused by Ménière's disease, vestibular neuritis or drug internal ear disorder. 37. The method of any one of claims 31 to 36, wherein the IL-6 antagonist is an antibody against an IL-6 receptor. The method of claim 37, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor. The method of claim 38, wherein the antibody against the IL-6 receptor is a monoclonal antibody against human IL-6 receptor. The method of claim 38, wherein the antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor. 41. The method of any one of claims 37-40, wherein the antibody against the IL-6 receptor is a recombinant antibody. The method of claim 39, wherein the monoclonal antibody against the human IL-6 receptor is a PM-1 antibody. The method of claim 40, wherein the monoclonal antibody against the mouse IL-6 receptor is an MR 16-1 antibody. The method of any one of claims 37-43, wherein the antibody against the IL-6 receptor is a chimeric antibody, humanized antibody or human antibody against the IL-6 receptor. 45. The method of claim 44, wherein the humanized antibody against the IL-6 receptor is a humanized PM-1 antibody.

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